Alternative splicing is a form of genetic regulation that enables the production of multiple proteins from a single gene. This study is one of the first to investigate variation in alternative splicing during a major evolutionary transition. We analyzed RNA from wild and domesticated sunflowers to examine differentiation in splice patterns during domestication. We identified divergent splice forms that may be involved in seed development, a major target of selection during domestication. Genetic mapping revealed that relatively few regulatory switches affecting many proteins have been altered in domesticated sunflowers. Our findings indicate that differences in splicing arose rapidly during a recent evolutionary transition and appear to contribute to adaptation and population divergence.