The Prolactin (PRL) hormone gene family shows considerable variation among placental mammals. Whereas there is a single PRL gene in humans that is expressed by the pituitary, there are an additional 22 genes in mice including the placental lactogens (PL) and Prolactin-related proteins (PLPs) whose expression is limited to the placenta. To understand the regulation and potential functions of these genes, we conducted a detailed temporal and spatial expression study in the placenta between embryonic days 7.5 and E18.5 in three genetic strains. Of the 22 PRL/PL genes examined, only minor differences were observed among strains of mice. We found that not one family member has the same expression pattern as another when both temporal and spatial data were examined. There was also no correlation in expression between genes that were most closely related or between adjacent genes in the PRL/PL locus. Bioinformatic analysis of upstream regulatory regions identified conserved combinations (modules) of putative transcription factor binding sites shared by genes expressed in the same trophoblast subtype, supporting the notion that local regulatory elements, rather than locus control regions, specify subtype-specific expression. Further diversification in expression was also detected as splice variants for several genes. In the present study, a detailed temporal and spatial placental expression map was generated for all murine PRL/PL family members from E7.5 to E18.5 of gestation in three genetic strains. This detailed analysis uncovered several new markers for some trophoblast cell types that will be useful for future analysis of placental structure in mutant mice with placental phenotypes. More importantly, several main conclusions about regulation of the locus are apparent. First, no two family members have the same expression pattern when both temporal and spatial data are examined. Second, most genes are expressed in multiple trophoblast cell subtypes though none were detected in the chorion, where trophoblast stem cells reside, or in syncytiotrophoblast of the labyrinth layer. Third, bioinformatic comparisons of upstream regulatory regions identified predicted transcription factor binding site modules that are shared by genes expressed in the same trophoblast subtype. Fourth, further diversification of gene products from the PRL/PL locus occurs through alternative splice isoforms for several genes.