South American cyprinodontiform fish are potential candidates to be used as model biomarker species of exposure in environmental toxicology. The aim of this study was to identify molecular and biochemical biomarkers of pollution using Poecilia vivipara (Poecilidae) and Jenynsia multidentata (Anablepidae). Partial nucleotide sequences for cytochrome P-450 1A (CYP1A), a classical biomarker of exposure to organic contaminants in fish, were identified in P. vivipara and J. multidentata (approximately 650 nucleotides) using degenerated primers and polymerase chain reaction (PCR). These sequences shared approximately 90% identity in the predicted amino acid sequence with the corresponding CYP1A region of Fundulus heteroclitus. Real-time quantitative PCR (RT-qPCR) analysis confirmed that CYP1A transcription was markedly induced in the liver and gills of J. multidentata (approximately185-fold and 20-fold, respectively) and P. vivipara (122-fold and 739-fold, respectively) 24 h after exposure to 1 μM synthetic CYP1A inducer β-naphthoflavone (BNF). At 24 h after injection with 1 μg/g environmental carcinogenic contaminant benzo[a]pyrene (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 fish did not produce changes in lipid peroxide (thiobarbituric acid-reactive substances, TBARS) levels, suggesting an absence of an oxidative stress condition. The newly identified CYP1A may thus serve as general biomarker of exposure to organic contaminant in future studies using P. vivipara and J. multidentata. Data also indicate the importance of species-specific differences in biomarker responses in these South American cyprinodontiform fish, suggesting distinct resistance/susceptibility properties to polycyclic aromatic hydrocarbons.