With Jadwiga's untimely death (1399), the dynastic connection between Lithuania and Poland was interrupted but restored when Jagiełło's son by a later marriage, Casimir IV, was chosen by Lithuanian boyars to be Grand Duke (r. 1440–1492) and then became King of Poland (r. 1447–1492). In the following decades, the Polish palatinate administrative structure, which had already been established in Galicia by 1440, was gradually introduced also in the Rus’-inhabited lands of Kyiv (1471), Volhynia (1566), and Bratslav (1566).

In these lands, nobles of the Catholic faith were favoured in significant ways over Eastern Orthodox Rus' nobles; but the latter rebelled (1430–1434) and won full political rights. In contrast, Orthodox Rus' leaders in Galicia experienced a continual decline in status under Polish rule. Previously politically powerful Orthodox boyars were forced to leave the area and were replaced by Polish officials, while gentry of the Catholic faith received large tracts of land. The Rus' leaders responded by forming alliances with Orthodox Moldavian leaders (1457–1504) and staging a rebellion. The rebellion (1490–1492), organized by local leader Petro Mukha, engaged nearly 10,000 Moldavian and Rus' peasants, as well as petty nobles and townspeople, in an unsuccessful attempt to overthrow Polish rule in southern Galicia.

In the mid-1500s, Muscovy's rulers began to act on their claim that they were the descendants of Riuryk, the declared founder of Kyivan Rus', and therefore the legitimate heirs of the territory of Kyivan Rus'. The threat from Muscovy and the acquisition by Ivan IV ("the Dread," r. 1547–1584) of several borderland strongholds from Lithuania (including Chernihiv) led to the second stage of Lithuania's increasingly closer association with Poland.