With the advent of the usage of primary fibroblasts in comparative and evolutionary biology, it is important for researchers to know the extent to which cells might be altered during the culturing process and how much species might differ in response to cell culture. We compared early changes in cell size and lipid composition of primary dermal fibroblasts grown at physiologically relevant oxygen concentrations (5% O2) from wild-caught species of birds. Fibroblasts from American Robins (Turdus migratorius L., 1766) and Bobwhite Quails (Colinus virginianus (L., 1758)) increased in size early in the culture process and cells from all five species of wild-caught birds exhibited changes in lipid-class composition. The two most common phospholipids, phosphatidylcholine and phosphatidylethanolamine, increased in concentration in all species between early passages and later passages of fibroblasts. Some less abundant lipid species, such as cardiolipin and sphingomyelin, exhibited similar concentrations in all three passages that we measured. Other lipid classes, such as cholesterol, increased in some species in later passages and decreased in others. Although results may vary with cell-culture conditions, this study points to a need for researchers comparing multiple species to take precautions when using cell culture, such as experimenting on the earliest possible passage of cells.