The accumulation of genetic incompatibilities between isolated populations is thought to lead to the evolution of intrinsic postzygotic isolation. The molecular basis for these mechanisms, however, remains poorly understood. The intertidal copepod Tigriopus californicus provides unique opportunities for addressing mechanistic questions regarding the early stages of speciation; hybrids between highly divergent populations are fertile and viable, but exhibit reduced fitness at the F2 or later generations. Given the current scarcity of genomic information in taxa at incipient stages of reproductive isolation, we utilize high-throughout 454 pyrosequencing to characterize a substantial fraction of protein-coding regions (the transcriptome) of T. californicus. Our sequencing effort was divided equally between two divergent populations in order to estimate levels of divergence and to reveal patterns of selection across the transcriptome. Assembly of sequences generated over 40 000 putatively unique transcripts (unigenes) for each population, 19 622 of which were orthologous between populations. BLAST searches of public databases determined protein identity and functional features for 15 402 and 12 670 unigenes, respectively. Based on rates of nonsynonymous and synonymous substitutions in 5897 interpopulation orthologs (those >150 bp and with at least 2X coverage), we identified 229 potential targets of positive selection. Many of these genes are predicted to be involved in several metabolic processes, and to function in hydrolase, peptidase and binding activities. The library of T. californicus coding regions, annotated with their predicted functions and level of divergence, will serve as an invaluable resource for elucidating molecular mechanisms underlying the early stages of speciation.