Variation of venom proteome is relevant to basic research, to management of envenoming, and to studies on the evolution of poisonous snakes. In this study, we explored the venom proteomes of eighteen Bothrops jararaca specimens of a single litter born and raised in laboratory. Using electrophoretic techniques and various protocols for measuring the proteolytic activities of these venoms we have detected individual variability and highlighted sex-specific proteomic similarities and differences among sibling snakes. SDS-polyacrylamide gel electrophoresis under non-reducing conditions showed protein bands of approximately 100 kDa specific of male venoms. 2D-electrophoresis showed regions with varying spot complexity between pooled female and male venoms as well as spots that were gender specific. Gelatin zymography showed that female venoms contained proteinases of approximately 25 kDa absent from male venoms. Female venoms were more active than male venoms in degrading fibrinogen whereas on fibrin no significant differences were detected. Among various chromogenic peptide substrates tested, male venoms showed higher amidolytic activity than female venoms on D-Val-Leu-Lys-pNA and D-Phe-Pip-Arg-pNA. Taken together, these results show sex-based differences in the venom proteome of sibling snakes of a single litter raised under controlled conditions which seem to be genetically inherited and imposed by evolutionary forces.