Crustacean hyperglycemic hormone (CHH) and vitellogenesis-inhibiting hormone (VIH), produced by the X organ–sinus gland neurosecretory complex, belong to a peptide group referred to as the CHH family, which is widely distributed in arthropods. In this study, genetic variants and post-translationally modified isoforms of CHH and VIH were characterized in the European lobster Homarus gammarus. With the use of RP-HPLC and ELISA with specific antibodies that discriminate between stereoisomers of CHH and VIH, two groups of CHH-immunoreactive peaks were characterized from HPLC fractions of sinus gland extract (CHH A and CHH B); each group contained two variants (CHH and D-Phe3CHH). In the same way, two VIH-immunoreactive peaks (VIH and D-Trp4VIH) were demonstrated in HPLC fractions from sinus gland extract. The masses of these different neuropeptides were determined by FT-ICR MS: CHH A and CHH B spectra exhibited monoisotopic ions at 8557.05 Da and 8527.04 Da, respectively, and both VIH isomers displayed an m/z value of 9129.19 Da. Two full-length cDNAs encoding preprohomones of CHH A and CHH B and only one cDNA for VIH precursor were cloned and sequenced from X organ RNA. Comparison of CHH sequences between European lobster and other Astacoidea suggests that the most hydrophobic form appeared first during crustacean evolution.