Spodoptera frugiperda is the most economically important maize pest in Brazil. There is little information about the genetic structure, using SSR markers, of S. frugiperda populations collected from maize crops. In this study, 21 SSR markers were used to evaluate the genetic diversity and population structure of S. frugiperda collected from distinct Brazilian geographical regions. Two hundred and twenty-seven alleles were recorded with an average of 10.76 per marker, and Polymorphic Information Content (PIC) values ranging from 0.242 to 0.933, with an average of 0.621, indicating a high discriminating power. The overall F ST , 0.061, indicated a moderate genetic differentiation among the S. frugiperda populations collected from maize, and the AMOVA showed that 87.36% of the genetic variation is within populations. The Mantel test showed significant correlation between genetic and geographic distances. The genetic data demonstrated that all individuals from the six sampling sites were structured as two sub-populations, being one of them composed only by the CL population, collected in the Rio Grande do Sul state. The knowledge about genetic diversity and population structure of S. frugiperda is important for the development of strategies for the insect pest management and monitoring systems, especially for the differentiated CL population.