The Balsas Basin is one of the largest and most varied formations in Mexico with a high degree of endemism and variety of flora and fauna. To date, there has been no in-depth study of spider diversity in this region. This paper details a survey of spiders found in the Balsas Basin, collected at 20 localities in the states of Guerrero, Puebla, and Morelos between 1997 and 2011. During our survey, we identified 437 mature spiders and grouped them into 24 families, 53 genera, and 56 species and sorted as follows: we found 15 species in Guerrero, 29 in Morelos, and 20 in Puebla. We found five new country records for Mexico and 28 new state records (nine for Guerrero, 10 for Morelos, and nine for Puebla), with a total of 13 possible new species. We estimated beta diversity (change in species composition), and indicated that most localities shared only a few species (fewer than 40%), which suggests that each locality has a unique suite of species with specific habitat characteristics. This first report on spiders from this extensive region shows the necessity for further study in the areas of biodiversity, ecology, and conservation biology.