Envenoming by scorpions in genusTityusis a public health problem in Tropical America. One of the most medically significant species isTityus trivittatus, which is known to occur from southwest Brazil to central-northern and eastern Argentina. In this work, we studied the lethality, composition, antigenicity, and enzymatic activity of venom from aT.trivittatuspopulation found further north in urban areas of eastern Paraguay, where it has caused serious envenomation of children. Our results indicate that the population is of medical importance as it produces a potently toxic venom with an LD₅₀around 1.19 mg/kg. Venom neutralization in preliminary mouse bioassays was complete when using Brazilian anti-T.serrulatusantivenom but only partial when using Argentinean anti-T.trivittatusantivenom. Venom competitive solid-phase enzyme immunoassays and immunoblotting from Argentinean and ParaguayanT.trivittatuspopulations indicated that antigenic differences exist across the species range. SDS-PAGE showed variations in type and relative amounts of venom proteins betweenT.trivitattussamples from Argentina and Paraguay. MALDI-TOF mass spectrometry indicated that while some sodium channel toxins are shared, including β-toxin Tt1g, others are population-specific. Proteolytic activity by zymography and peptide identification through nESI-MS/MS also point out that population-specific proteases may exist inT.trivitattus, which are postulated to be involved in the envenoming process. A time-calibrated molecular phylogeny of mitochondrial COI sequences revealed a significant (8.14%) genetic differentiation between the Argentinean and Paraguayan populations, which appeared to have diverged between the mid Miocene and early Pliocene. Altogether, toxinological and genetic evidence indicate thatT.trivitattuspopulations from Paraguay and Argentina correspond to distinct, unique cryptic species, and suggest that further venom and taxonomic diversity exists in synanthropic southern South AmericanTityusthan previously thought.