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Hairy marron (Cherax tenuimanus Smith) are critically endangered freshwater crayfish found only in a single river in south-west Australia. Conservation efforts have included a captive breeding program, which has been largely unsuccessful, despite the closely related smooth marron (Cherax cainii Austin) being successfully bred for aquaculture. Using an untargeted liquid chromatography-mass spectrometry (LC-MS) metabolomic approach we created a profile of the metabolites in the haemolymph for males and females of the two species of marron. A non-lethal method was used to collect haemolymph and 84 reproducible annotated metabolites were identified. Variation in the levels of some metabolites were detected between species and between sexes within species. Multivariate analyses clearly differentiated the congeneric species and univariate analyses identified differences between species, sex and for some metabolite interactions between species and sex. This study created a baseline metabolome dataset for the two species and began to investigate the biological significance of metabolites that varied between species. We have shown metabolomics could be used for targeted studies to potentially assist reproductive success. This approach will be beneficial for conservation and aquaculture practices with potential applications for other aquatic taxa worldwide.