In the nematode genus Caenorhabditis, most species are gonochoristic (male/female), and several hermaphrodite species (male/hermaphrodite), including C. elegans, are thought to have evolved independently from gonochoristic species. In this study, we compared the sex determination pathways between C. elegans (hermaphrodite species) and its closest species, C. inopinata (gonochoristic species), to elucidate the genomic changes responsible for the differences in their reproductive modes. Based on the comparison of genome sequences, we found three significant differences in the sex determination pathway genes in C. inopinata. 1)
xol-1, which is on Chr. X and responsible for the male sex determination in C. elegans, is duplicated and localize on Chr. I and Chr. X, but the latter is disrupted by transposon insertion. 2) A transposon insertion disrupts the coding region of
her-1, which is essential for the male development in C. elegans. 3)
fog-2, which is essential for temporal masculinization in C. elegans hermaphrodite germline, is absent in C. inopinata. Next, to investigate functional conservation of the sex determination genes, each of their expression in C. inopinata was inhibited by RNAi, and sex determination phenotypes were analyzed. Similar to C. elegans, RNAi-knockdown of
tra-2 and
tra-1 caused masculinization of C. inopinata female germline. However, knockdown of more upstream genes involved in both sex determination and dosage compensation (
sex-1,
xol-1,
sdc-2) and genes involved in male sex determination (
her-1,
fem-1) did not show any detectable phenotypes, suggesting that these genes do not play significant roles for sex determination in C. inopinata. These results suggest that the sex determination pathways in C. elegans and C. inopinata are surprisingly different from each other, and only the downstream part, including
tra-2 and
tra-1, seems to be conserved; in C. inopinata, an alternative set of genes is likely to regulate the early male/female decision and dosage compensation.