Genetic variability within the Suwannee River, Florida, population of Gulf of Mexico sturgeon, Acipenser oxyrhynchus desotoi, was assessed by examining sequence and length variation within the control region, or D-loop, of the mitochondrial genome. Although once abundant throughout the Gulf of Mexico, Gulf sturgeon are now listed as a threatened species by the U.S. Fish and Wildlife Service. Mitochondrial DNA was analyzed for length variation from 168 individual Gulf sturgeon by PCR amplification and visualization of PCR products using ethidium bromide-stained agarose gels. Of the 168 individual Gulf sturgeon, 31 (18.5%) were heteroplasmic for one to four copies of an 81-base pair, tandemly repeated sequence in the D-loop region. However, no individuals homoplasmic for multiple copies of the repeat sequence were observed. The existence and nature of these tandem repeats in heteroplasmic individuals was confirmed by direct sequencing of the PCR products for a subset of 22 individuals. The results are consistent with the apparent nature and mechanism of heteroplasmy observed in a congeneric species, A. transmontanus. In addition, sequences for 187 base pairs outside of the tandem repeats were identical among all 16 individuals assayed for this region. Lack of variable sequences is concordant with earlier studies involving mtDNA restriction fragment length profiles of Gulf sturgeon found in the Suwannee River. The absence of sequence variation exclusive of the tandem repeats is consistent with the hypothesis that the subspecies has undergone a population or evolutionary bottleneck.