Two full-length cDNA (Pt-CHH1 and Pt-CHH2) sequences encoding crustacean hyperglycemic hormone (CHH) were cloned from tissues of the swimming crab (Portunus trituberculatus) using RACE. Pt-CHH1 was cloned from eyestalk, whereas Pt-CHH2 was cloned from thoracic ganglia. Sequence and structure analyses of Pt-CHH1 and Pt-CHH2 suggest that they may be generated from alternative splicing. Tissue distribution showed that transcript of Pt-CHH1 was only detected in eyestalk, while transcript of Pt-CHH2 was observed in several extra-eyestalk tissues. The transcript levels of Pt-CHH1 and Pt-CHH2 during molting and ovarian development were determined using qPCR. In molting process, level of Pt-CHH1 in eyestalk increased from stage A (postmolt), and to significant higher at stage C (intermolt), then decreased during premolt (D0–D4). In ovarian development, level of Pt-CHH1 in eyestalk decreased from previtellogenic stage (II), and to significant lower at mature stage (IV). The expression patterns of Pt-CHH2 in thoracic ganglia and Y-organ were distinct from that of Pt-CHH1 in eyestalk. The combined results suggest that Pt-CHH1 may be involved in inhibition of molting and ovarian development, whereas Pt-CHH2 may have other physiological functions.