Temperature-dependent sex determination is a notable model of phenotypic plasticity; however, its underlying molecular mechanism remains unclear.As a typical TSD species, the red-eared slider turtle (Trachemys scripta) has its offspring gender completely determined by incubation temperature. In order to provide more basic data for illustrating the TSD mechanism, transcriptome sequencing was performed on T. scritpa early embryonic gonads of stage 16 at different temperature (male-producing temperature (MPT) and female-producing temperature(FPT)) in this study. IlluminaHiseq 2500 high-throughput platform was used for transcriptome sequencing. A total of 474 differentially expressed genes were screened out, among which 283 genes showed MPT higher expression and 191 genes showed FPT higher expression. By comparing with NR, Swiss-Prot, GO, COG, KOG and KEGG databases, 60 GO functional annotation classifications and 139 KEGG metabolic pathways were obtained. According to functional annotation of differentially expressed genes, these candidates included:sex determination and differentiation related conserved genes, such as Dmrt1, Sox9, Foxl2, etc; steroid hormone metabolism related genes, such as Cyp11b1, Hsd11b2, etc; heat-sensitive protein coding genes, such as Hspb6, Dnaja4, Serpinh1, etc. Several selected candidates were verified by quantitative PCR and immunofluorescence. In conclusion, some new temperature sensitive and hormone metabolism related factors were found out from RNA-seq data of turtle embryonic gonads, providing basic support for illustration of the molecular mechanism of TSD.