Chrysodeixis includens (Walker, 1858) and Rachiplusia nu (Guenée, 1852) are important defoliating pests belonging to the Plusiinae subfamily in the Western hemisphere. C. includens is a major lepidopteran pest of soybean in the Americas, whereas, until 2021, R. nu was more restricted to the temperate regions of South America. Recently, reports of R. nu feeding on Cry1Ac soybean and occurring in tropical regions of Brazil have raised questions regarding the distribution of this species. The morphological similarity of the larvae from the two species makes it difficult to correctly identify the species in the field, which may lead to an underestimation of R. nu in Brazilian territory. This study aimed to address these questions by using a molecular approach to identify Plusiinae caterpillars throughout three seasons in non-Bt, Cry1Ac, and Cry1Ac × Cry1F soybean fields. Here, we carried out a comprehensive spatial sampling of the primary soybean-producing regions in Brazil. The results showed that R. nu has been the main Plusiinae occurring in soybean over the last three years, and it was present in all sampled regions. For Cry1Ac and Cry1Ac × Cry1F soybeans, up to 99% of the samples collected in 2023/24 were identified as R. nu. Non-Bt soybeans had higher variations in the proportion of the two species among the regions and across seasons, indicating that populations of C. includens and R. nu are co-occurring throughout the country. This is, to our knowledge, the most robust report assessing the distribution of C. includens and R. nu in Brazil using a molecular tool. This study provides clarification of R. nu occurrence and highlights the importance of pest monitoring from an integrated pest management perspective.