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Nocturnal Lepidoptera represent a highly diverse and ecologically important insect group and are widely used in biodiversity assessments due to their sensitivity to habitat conditions. This study aimed to compare the diversity, structure, and species composition of nocturnal Lepidoptera in two rural localities of Panama: La Ocho (Tortí, Chepo District) and Arenal (Río Congo Arriba, Santa Fe District). Nocturnal sampling was conducted between June and August 2019 using light attraction with white and blue light sources directed onto a white sheet. Diversity was evaluated using the Shannon–Wiener and Simpson indices, and similarity between sites was assessed using the Jaccard coefficient. Individual-based rarefaction curves were used to standardize sampling effort and compare expected species richness between localities. Species richness and diversity were higher at the Arenal site, which showed greater Shannon diversity and lower dominance values compared to La Ocho. Species composition differed markedly between sites, as reflected by a low Jaccard similarity coefficient. Rarefaction analyses supported these patterns, indicating higher expected species richness at Arenal across comparable sampling effort. These results highlight the influence of habitat conditions on nocturnal Lepidoptera assemblages and support the usefulness of moths as ecological indicators in tropical rural environments.