At least 25,000 playa wetlands exist in the Southern Great Plains, and most are embedded in cropland-dominated watersheds, resulting in sediment-induced alterations in hydrology and potential contamination from agricultural chemicals. These stressors have been hypothesized or shown experimentally to affect the development and function of the immune system of amphibians. We hypothesized an association between land use and immune function in playa amphibians. We compared body mass index (body mass/SVL), spleen mass index (spleen mass/SVL), and splenocyte count index (splenocyte count/SVL) in three developmental stages (tadpoles, metamorphs, juveniles) of Spea bombifrons and Spea multiplicata collected from playas embedded in either cropland or native grassland watersheds. Body mass indices of S. bombifrons and S. multiplicata tadpoles collected from grassland playas were 39% and 29% greater, respectively, than those from cropland playas. Similar responses were observed for spleen mass and cellularity in both species, as spleen mass indices and spleen cellularity indices of S. bombifrons tadpoles from grassland playas were 330% and 1,000% greater, respectively, than for tadpoles collected from cropland playas. Spleen mass and cellularity of S. multiplicata also were influenced by land use, but unlike S. bombifrons, differences were of a lower magnitude and more consistent across developmental stages. This is the first field study to link habitat alteration with altered immune system development in amphibians.