Reliable individual identification is fundamental to ecological and behavioural studies, yet many marking techniques are invasive and may affect animal behaviour or survival. Biometric identification approaches offer a non-invasive alternative for individual recognition using phenotypic traits. In amphibians, body patterns, especially colouration, have been used to identify individuals, but species lacking permanent body patterns require alternative traits. This study evaluated whether iris patterns could serve as a marker for individual recognition in the treefrog Osteocephalus oophagus. A capture-recapture study was conducted over three breeding seasons (2021–2023) in French Guiana. At each encounter, eye photographs were compared with an image library to identify individuals without handling. Manual identifications were independently verified using Wild-ID software. Across 392 capture events involving 103 individuals, iris patterns were highly distinctive among individuals and enabled repeated identification across encounters. Within-season recapture rates ranged from 56% to 80%, indicating that iris patterns remained sufficiently stable for short-term monitoring. However, only three individuals were re-identified across years, preventing a robust assessment of long-term pattern stability. As these individuals showed no substantial alteration in iris patterns, the low interannual recapture rate was more likely explained by population turnover or emigration than by changes in iris morphology. The findings suggest that iris recognition provides an effective, non-invasive method for short-term individual identification in O. oophagus, reducing the need for repeated handling and supporting ethical wildlife research. Further studies are needed to assess the long-term stability of iris patterns and evaluate the applicability of this method to other amphibian species.