Chemical signaling in animals often plays a central role in eliciting a variety of responses during reproductive interactions between males and females. One of the best-known vertebrate courtship pheromone systems is sodefrin precursor-like factors (SPFs), a family of two-domain three-finger proteins with a female-receptivity enhancing function, currently only known from salamanders. The oldest divergence between active components in a single salamander species dates back to the Late Paleozoic, indicating that these proteins potentially gained a pheromone function earlier in amphibian evolution. Here, we combined whole transcriptome sequencing, proteomics, histology, and molecular phylogenetics in a comparative approach to investigate SPF occurrence in male breeding glands across the evolutionary tree of anurans (frogs and toads). Our study shows that multiple families of both terrestrially and aquatically reproducing frogs have substantially increased expression levels of SPFs in male breeding glands. This suggests that multiple anuran lineages make use of SPFs to complement acoustic and visual sexual signaling during courtship. Comparative analyses show that anurans independently recruited these proteins each time the gland location on the male’s body allowed efficient transmission of the secretion to the female’s nares.