The first years of the Peloponnesian war, Chios remained loyal to the Athenians. The verses of Eypoledos, in the play “Cities”, which was put up in the Athenian theatre, and was written around 420 B.C., mentioned about the relations of Chios and Athens: "Chios, the beautiful city, which sends us martial ships and men when it is needed, but it also complies to the rules without ever arguing, just like a horse does without any whips."

The first martial mission, which the fleet of Chios participated in, was during the expedition of Pericles in Argolida (around 430 B.C.). Chios and Lesvos sent fifty triremes. In 425 B.C. four ships from Chios helped the Athenians in the victorious expedition of Pilos and in 423 B.C. ten ships from Chios reinforced Nikias’ fleet in Halkidiki. Chios, by sending enough ships, also participated in the expedition of the Athenians in Dorian Milos (416 B.C.), which was destroyed by their army.

The islands of Chios and Lesvos sent about fifty-five boats to help the Athenians in their expedition in Sicily (415 B.C.). The Athenians realised that they could not occupy Syracuse and they decided to return to Greece. However, Syracusians occupied the outlet of the Big Harbour and they made it impossible for their enemy to leave the place. Thus Athenians abandoned their ships and they disembarked. The Syracusians arrested them and most of them were made to work in the Quarries. After seventy days they were sold as slaves. Very few people from Chios survived and managed to bring the terrible news to their homeland.

After the expedition in Sicily, Athens had lost its great power force and it began to lose also its allies. Thus Chios, in 412 B.C., was from the first allies to abandon the alliance, after having been loyal to the Athenian alliance for sixty-six years. The Athenians were alarmed a lot by the apostasy of Chios, their strongest ally among the Ionian cities. So they sent twenty triremesin order to suppress the rebellion. Chios tried to ask for help from the other allies, like Miletus, Lesvos, Mithimna in order to confront effectively the Athenians, and allied with Lacedaemon, from whom they also asked for help. First, the Athenians attacked the countryside of Chios, disembarking in Volissos and Kardamyla, where they had a glorious victory and killed many citizens of Chios. The Chians were also defeated in the battles in Fana and Leykonio, and then they barricaded themselves in the city’s Walls. Later, Pedaritos from Sparta with five hundred mercenaries reached Chios, and found the island in a terrible situation. Mercenaries of Pedaritos were turned away by the Athenians and Pedaritos was killed. The Athenians besieged the city of Chios, which was attacked by famine. Sparta, the ally of Chios, did not send any help this time. Thus the Chians decided not to wait any longer for Sparta to send any help and they rebelled against the Athenians counting only on their forces.  They attacked the thirty-two Athenian ships with thirty-six boats and they had a glorious victory.

In 405 B.C., the contribution of Chios fleet in the naval battle in the Aigos Rivers  was very important since that battle played a decisive role in the end of the war and it brought an end to the most glorious period of Athens. The leaders of Chios fleet were honoured with statues made by Lysander and his collaborators. Isocratescommenting the events says that, "What I would say about Chios is that whichever from the two opponents gains its support, this will also be the one who will be the dominant of the sea". This phrase shows the important role of Chios in that period.
During the next years, Chios remained in the alliance with Sparta and helped with its fleet, until the end of the war (404 B.C.).