Evolutionary biology often seeks to decipher the drivers of speciation. Much debate persists over the relative importance of isolation and gene flow in the formation of new species. Genetic studies of closely related species can assess if gene flow was present during speciation because signatures of past introgression often persist in the genome. We test hypotheses on which mechanisms of speciation drove diversity among three distinct lineages of desert tortoise in the genus Gopherus and where different biogeographic patterns (vicariance vs. ecological segregation) are observed at opposing ends of their distributions. We performed RNAseq using nine individuals in three flowcell lanes on the Illumina HiSeq platform. We dedicated two lanes to high-coverage sequencing of three individuals, one for each of the following lineages: G. agassizii was from a captive individual in Arizona that originated in California (Moj_A haplotype); Sonoran lineage G. morafkai was collected from a captive individual from Arizona; and Sinaloan lineage G. morafkai was obtained from just outside of Alamos, Sonora Mexico (Rancho Las Cabras; RLC). Data from these individuals were used to assemble reference transcriptomes. We used the third flowcell lane to generate low-coverage RNA-seq reads from six samples that were then mapped to the reference assembly to assess diversity within and among the three lineages. For these six samples, we hand-captured and collected samples from wild desert tortoises from the following three sites in 2013: two Sinaloan G. morafkai from the Reserva La Sierrita, Sierra de Alamos, Sonora, Mexico (tropical deciduous forest); two Sonoran G. morafkai from the Rancho San Judas north of Hermosillo, Sonora, Mexico (Sonoran desertscrub/plains of Sonora); and two G. agassizii from Trout Canyon west of Las Vegas bordering the Eastern Mojave and Northeastern Mojave recovery units, Clark County, Nevada (Mojave desertscrub). We characterized 20,126 synonymous variants from 7,665 contigs called in six individuals, representing each of the three lineages. We analyzed RNA-seq data using the diffusion approximation for demographic inference algorithm in the software package ?a?i to test the null hypothesis of no admixture during divergence. The best-fit model in the ?a?i analysis for the relationship among the three taxa is concordant with the species tree. The ?a?i analysis failed to demonstrate evidence of gene flow during divergence among any of the three lineages. These analyses suggest that divergence among the lineages occurred in the absence of gene flow, whether through physical allopatry or ecological niche segregation. Our results validate species-level differentiation among the three lineages of desert tortoise.