Morphologically distinct males and females are observed throughout the animal kingdom. Why and how sex evolved and is maintained in most living organisms remains a key question in cellular and evolutionary biology. Here we report that four isoforms of dmrt1 (dsx- and mab3-related transcription factor 1) are generated in testis, ovotestis, and ovary by alternative splicing in the rice field eel, a fresh water fish that undergoes natural sex reversal from female to male during its life cycle. These transcripts encode four different size proteins with 301, 196, 300, and 205 amino acids. Like fly doublesex splicing, the dmrt1 of the rice field eel is also alternatively spliced at the 3′ region, which generates diverse isoforms in gonads by alternative use of 3′ sequences. Not only is dmrt1 expressed specifically in gonads, but its multiple isoforms are differentially coexpressed in gonadal epithelium during gonad transformation. Expression levels of a and b isoforms of dmrt1 ranged from low to high (ovary < ovotestis I < ovotestis II < ovotestis III < testis), based on comparisons of mean values from real-time fluorescent quantitative reverse transcription-polymerase chain reaction analysis. The overall expression level of dmrt1 b was much lower than that of dmrt1 a. Expression of dmrt1 d was not only low, but it also did not change significantly during sex transformation. The differential expression of dmrt1 isoforms may also be regulated by their 3′ untranslated regions (UTRs), although these 3′ UTRs do not contribute to intracellular localization of the Dmrt1 protein. These results provide new insight into roles of regulation at the level of splicing of dmrt1 in governing the sex differentiation cascade.