The main themes of Albion's Seed are that each of the four major English-speaking cultural groups that settled British America in the 17th and 18th centuries brought complementary, though often contrasting, folkways reflecting their different social, cultural, and religious origins. How these group's cultures and ideas persisted in their respective regions and then spread Westward has profoundly influenced American social, political, and economic norms, according to the author. This ethnographic approach disaggregates the labels "English" or "British" and drills down into the often widely divergent attitudes and beliefs these different groups of settlers had on dozens of central issues. The overarching issue of their understanding of liberty is a dominant thread uniting the narrative.

The Protestant Reformation divided Europe. Catholic-on-Protestant and Protestant-on-Catholic violence peaked in the first century of English settlement in America. Tens of thousands of English Protestants in England's densest urban area to the northeast of London decided to move to America to create their ideal society away from disapproving officials. They were roughly twice as literate as the English average and came from a region noted for its outsized contributions to English intellectual life. Many of its leaders were educated at Cambridge University, where some of them had been college fellows and deans. They brought an idea of ordered liberty, by which they meant the liberty to do good but not the liberty to do evil. They imposed these restraints on themselves as a public society and did not believe they could or should be imposed from without as long as they remained within English law and their colonial charters.

The Virginian idea of hegemonic liberty was brought by Governor Sir William Berkeley and his distressed Cavaliers to the Old Dominion. These Royalists in the 17th century believed their high estate brought them greater liberty than those of low estate, and they believed that their right to rule followed from this. This cultural comfort with hierarchy made slavery a natural outcome in Virginia. At the time, it was believed that anyone that was not independent in wealth could not help but be self-interested in government. This was thought to lead to corruption, as self-interested men would seek to line their pockets through their public offices. By the 18th century, these men and their counterparts in England had become Whigs, and their conception of liberty fell in line with classical Lockean liberalism, especially the Lockean beliefs about property rights. The state had to be small not to infringe on property rights, and the rule of law was paramount. Eventually, the hegemonic conception of ownership of self was extended downward until everyone was ostensibly included in the ruling class through the franchise. It remained until after the Civil War, and several Constitutional amendments were created for these rights to be extended to former slaves.

The Quakers of the Delaware Valley came from the opposite end of the social scale (they were mostly of the yeomanry) and believed that any right reserved to themselves should be extended to all (apart from reading, as they had doubts about its benefit for the working class). By the 18th century, they had become prosperous and had dropped the more extreme aspects of their religion while holding onto the better parts, such as freedom of conscience. Their liberal approach to rights influenced the founding fathers during the revolution and the Constitutional convention, when the Quakers hosted British colonial leaders in their capital city of Philadelphia.

The backcountry settlers of the upland rural south were ultimately the largest wave of early British settlers coming from the northern English and lowland Scottish border region, as well as from Protestant northern Ireland. Although generally poor, they were fiercely proud and independent. They brought their clan system and values of raids, retaliation, and blood feuds ,making them perhaps the most warlike of the four groups. Their definition of liberty was non-reciprocal and amounted to being left alone, subject to their own elites who were known in the border regions of Britain as 'the ascendancy'. To maintain their own independence, they had to join with the other three groups in the revolution. They were natural frontiersmen like Daniel Boone, and their military prowess has led to their outsized representation in America's armed forces since the 18th century.

Forced to work together against a common adversary in the revolution, these four groups hammered out a conception of liberty that has, in the view of the author, come to be one of America's greatest legacies to the world.