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As with most crimes, the killer becomes a suspect in the minds of those around him.
Macduff suspects Macbeth's activities immediately after the discovery of the body. Macbeth admits to killing the guards which makes Macduff suspicious enough to ask
Later, Macduff refuses to attend Macbeth's coronotion worrying that the new direction of the kingdom is evil, as evidenced by his quote from scene iv
The King's sons flee the scene, afraid for their own live. They sense that someone close to their father killed him when Donalbain says in scene iii
The Thane of Ross also questions Duncan's sons as suspects because they had really nothing additional to gain from the murder, especially if they ran away! He says in scene iv
Ross, Donalbain and, most importantly, Macduff suspect Macbeth in Act II.
Donalbain and Malcolm decide to flee when they find that their father has been murdered. They even decide to go separate ways. This is a quote from Donalbain in Act II, sc 3.
He knows that someone close to Duncan was the murderer--as said in his last 2 lines.
However, in Act III, Banquo has a better quote that proves he suspects Macbeth.
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