BACKGROUND: Snakes of the genus , popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, and deserve special attention due to the severity of their bites and for inhabiting densely populated areas. Regarding the treatment of snakebites by , questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans. Thus, the development of a differential diagnostic kit for this species would be of great interest because it provides, for healthcare professionals, a tool that would allow us to determine whether the accident was caused by or other species of the genus. It would also make it possible to evaluate the specificity of the treatment and to provide data for epidemiological studies. METHODS: First, we produced a species-specific polyclonal antibody - a potential biomarker of venom - against bothropstoxin-I (BthTx-I), which is also found in smaller quantities in the venoms of from southern Brazil. RESULTS: Polyclonal antibodies against bothropstoxin-I could be separated into several species-specific immunoglobulins. Then, aiming to develop a system of safe and standardized immunoassay, we produced monoclonal antibodies. Seven hybridomas were obtained. Five of them were specific to the venom of and two recognized the venom of from the southeastern population. The use of monoclonal antibodies also made it possible to differentiate from venom obtained from the southern population. Analyzing the reactivity of monoclonal antibodies against other bothropic venoms, we found mAb Bt-3 to be more specific than others for venom. CONCLUSIONS: These results show the potential of BthTx-I for producing monoclonal antibodies that differentiate between and other species venoms.