We used mitochondrial 16S ribosomal DNA (16S rDNA) and microsatellites to study genetic diversity and population structure of gray sunflower seed weevil (Smicronyx sordidus L.). A 400-bp fragment of mitochondrial 16S rDNA was amplified and sequenced from weevils collected from 20 geographic populations. Thirty-three unique mitochondrial DNA sequences (haplotypes) were identified based on 62 polymorphic sites. Sequence divergence among haplotypes ranged from 0.2 to 3.68%. Four of 33 haplotypes were shared among populations, but most (88%) were found in only one population. No geographical disjunctions could be correlated with cladogenesis shown by phylogenetic reconstruction of unique haplotypes. Haplotype 33 from Texas separated from a major clade composed of other gray and red sunflower seed weevil haplotypes. AMOVA results revealed that most of the genetic variation observed in gray sunflower seed weevil was due to variation within populations. Nested clade analysis showed two main clades with significant geographic structure. Contiguous range expansion appeared to be the main mechanism of increased variation within the species. Significant differences were found in analyses of five polymorphic microsatellite loci in four geographically separated field populations from which multiple individuals could be collected from wild sunflowers. More genetic divergences occurred among weevil populations collected from sunflowers along the East-West axis than among weevil populations collected on a North-South axis. The overall results are consistent with current theories concerning the origin of the cultivated sunflower in central United States. The population represented by haplotype 33 from Texas is likely a new species that evolved independently.