South American Cyprinodontiform fishes are potential candidates to be used as model species in environmental toxicology. We sought for molecular and biochemical biomarkers of pollution in Poecilia vivipara (Poecilidae) and Jenynsia multidentata (Anablepidae). Partial nucleotide sequences for the cytochrome P450 1A ( cyp1A ), a classical biomarker of exposure to organic contaminants in fish, were identified in P. vivipara and J. multidentata (∼ 650 nucleotides) using degenerated primers and PCR. These sequences shared ∼ 90 % identity in the predicted amino acid sequence with the corresponding Cyp1A region of Fundulus heteroclitus . RT-qPCR analysis confirmed that cyp1A transcription was strongly induced in the liver and gills of J. multidentata (∼185-fold and ∼20-fold, respectively) and P. vivipara (122-fold and 739-fold, respectively), after 24-hrs exposure to 1 μM of the synthetic cyp1A inducer β-naphthoflavone (BNF). After 24 hs of injection with 1 μg.g -1 of the environmental carcinogenic contaminant benzoapyrene (BaP), a decreased total antioxidant capacity against peroxyl radicals was observed both in liver of J. multidentata and gills of P. vivipara . BaP injection in both fishes did not cause changes in lipid peroxides (TBARS) levels, suggesting an absence of an oxidative stress situation caused by BaP injection in this study. The newly identified cyp1As would serve as general biomarkers of exposure to organic contaminant in future studies using P. vivipara and J. multidentata . The results also points out to the important species-specific differences in the biomarker responses in those South American cyprinodontiform fishes, which would suggests distinct resistance/susceptibility to polycyclic aromatic hydrocarbons.