Expressed sequence tag analysis of normal and subtractive complementary DNA (cDNA) libraries of Thai abalone (Haliotis asinina Linneaus) was carried out. Several reproduction-related transcripts—for example, vitelline envelope zona pellucida domain 2 (Ha-VEZPD2), vitellogenin (Ha-VTG1), sperm lysin (Ha-SL), fertilization protein (Ha-FP), tektin A1 (Ha-TekA1), axonemal protein 66.0 (Ha-Axp66.0), and double-sex male abnormal-3-related transcription factor 1—(Ha-DMRT1)—were identified. The full-length cDNA of Ha-SL (665 bp containing an open reading frame (ORF) of 453 bp corresponding to a polypeptide of 150 amino acids) was obtained. In addition, that of Ha-VEZPD2.1 (1,094 bp with an ORF of 942 bp, 313 aa) and Ha-VEZPD2.7 (1,130 bp with an ORF of 1,002 bp, 333 aa) were also deduced after cluster analyses of ESTs encoding Ha-VEZPD2. Tissue distribution analysis indicated that Ha-Axp66.0 and Ha-DMRT1 were detected in testes but not in other tissues. Conversely, Ha-VEZPD2.1, Ha-VEZPD2.3, Ha-VEZPD2.17, Ha-VEZPD2.18, and Ha-VTG1 were expressed in ovaries but not in other tissues of male and female broodstock. Expression of these sex-related transcripts during gonad development was estimated by semiquantitative reverse transcription polymerase chain reaction. Relative expression levels of Ha-Axp66.0, Ha-TekA1, and Ha-DMRT1 in testes were significantly higher in stages II, III, and IV of testicular development (P < 0.05). In contrast, Ha-SL and Ha-FP levels were not significantly different during testicular development of H. asinina (P > 0.05). The expression levels of Ha-VEZPD2.1, Ha-VEZPD2.2, Ha-VEZPD2.3, Ha-VEZPD2.7, Ha-VEZPD2.17, Ha-VEZPD2.18, and Ha-VTG1 during stage III were significantly lower than those in stage I ovaries of H. asinina (P < 0.05).