Matrotrophic fish in the genus Poeciliopsis (Poeciliidae) have a placenta-like structure used in post-fertilization maternal provisioning of the developing embryo. To understand better the structure and function of the Poeciliopsis placenta we derived cDNA libraries from the maternal follicular placenta of two matrotrophic Poeciliopsis sister species, P. turneri and P. presidionis. These species inherited their placenta from a common ancestor and represent one of three independent origins of placentas in Poeciliopsis. Expressed sequence tags were generated and putative function was determined using BLASTX homology searches and Gene Ontology annotation. Reverse transcriptase-PCR was used to verify placenta tissue expression of a putative candidate gene, alpha-2 macroglobulin. 1956 (71.5% of the total submitted ESTs) and 924 (71.0% of the total submitted ESTs) unique transcripts were identified for the P. turneri and P. presidionis placenta, respectively. Homology search and Gene Ontology annotation revealed putative genes whose products may be involved in specific transport functions of the maternal follicle. These putative genes are excellent candidates for future research on the evolution of the placenta. We discuss our results in light of the parent-offspring conflict theory of placental evolution and in terms of the Poeciliid placenta structure and function.