The Mississippian Strathclyde Group of the Midland Valley of Scotland yields some of the earliest non-marine ostracods. The succession records shallow marine, deltaic, estuarine, lagoonal, lacustrine, fluvial and swamp environments representing a series of staging-posts between fully marine and limnetic settings. Macrofossils and ostracods are assigned to marine, marginal marine, brackish and freshwater environments based on their faunal assemblage patterns. Key brackish to freshwater ostracods are Geisina arcuata, Paraparchites circularis n. sp., Shemonaella ornata n. sp. and Silenites sp. A, associated with the bivalves Anthraconaia, Carbonicola, Cardiopteridium, Curvirimula, Naiadites, the microconchid ‘Spirorbis’, Spinicaudata and fish. Many Platycopina and Paraparchiticopina ostracods are interpreted as euryhaline, which corresponds with their occurrence in marine to coastal plain water bodies, and supports the ‘estuary effect’ hypothesis of non-marine colonization. The success of non-marine colonization by ostracods was dependent on the intrinsic adaptations of ostracod species to lower salinities, such as new reproductive strategies and the timing of extrinsic mechanisms to drive non-marine colonization, such as sea-level change. The genus Carbonita is the oldest and most common freshwater ostracod, and went on to dominate freshwater environments in the Late Palaeozoic.