Assessing genetic diversity and population structure is prerequisite to the systematic utilization and conservation of germplasm resources of crop plants. The genetic diversity and population structure in a combined panel of 557 Gossypium hirsutum L. accessions including 375 cultivars and 182 race stocks using 114 pairs of simple sequence repeat primers were evaluated in the current study. Six G. barbadense L. accessions were included as an out-group. Genotyping the diversity panel of 563 accessions with the markers identified a total of 819 alleles and 662 alleles within G. hirsutum. Population structure analysis identified one G. barbadense group and five G. hirsutum groups corresponding to southwestern cultivars, Mexican collections, western cultivars, southeastern and mid-south cultivars, and Guatemalan collections. Average genetic distance of 0.253 indicated a moderate level of genetic diversity in this panel. Analysis of molecular variance revealed a low level of differentiation among cultivated cotton groups compared to landrace accessions. Genetic diversity and population structure analyses suggest landraces of Guatemala could be a potential source of novel genetic variability for U.S. cotton. Further, multiple core sets with different levels of allele richness were identified. The diversity panel and the core sets identified could be a good resource for broadening the genetic base of U.S. cotton and for genetic analysis of agronomic traits.