Exposure to estrogens during the period of sexual differentiation is known to adversely affect the development of testes in African clawed frogs (Xenopus laevis), but little is known about molecular changes that coincide with the development of altered phenotypes. Therefore, the transcriptome-level effects of exposure to 17α-ethynylestradiol (EE2) during sexual differentiation of X. laevis were evaluated by use of Illumina sequencing coupled with RNA-Seq expression analysis. Overall, a number of processes were affected by 17α-ethynylestradiol, including steroid biosynthesis, thyroid hormone signaling and metabolism, testicular development, and spermatogenesis. Some of the altered pathways, such as thyroid hormone signaling and testicular development, could be linked with biological effects on metamorphosis and gonadal phenotypes, respectively, that were observed in frogs that were exposed to 17α-ethynylestradiol throughout metamorphosis and the early postmetamorphic period. Thus, early changes at the transcriptome-level were predictive of pathologies that did not manifest until later in development. To validate the quantitative capacity of RNA-Seq, a subset of transcripts identified to have altered abundances in individuals exposed to 17α-ethynylestradiol was also evaluated by use of quantitative polymerase chain reaction (qPCR). While small sample sizes (n = 3) limited the ability to draw conclusions pertaining to differences in qPCR-derived abundances of transcripts between control and exposed tadpoles, there was a significant relationship (r(2) = 0.78) between fold-changes for RNA-Seq and qPCR.