To assess genetic diversity in North American captive Asian elephants Elephas maximus, one mitochondrial DNA (mtDNA) segment was sequenced in combination with multilocus genotypes generated from 20 nuclear microsatellite loci for 201 individuals. The analysis of 627 bp of the C-terminal of cyt b and the hypervariable left domain of the noncoding control region (labeled as MDL fragment) sequences revealed the existence of two mtDNA lineages (α and β clade). Analysis of the MDL confirmed that North American captive Asian elephants belong to either the previously characterized α or β clade. An average nucleotide diversity of 0.017 was observed for the Asian elephant mtDNA MDL fragment sequences. Regardless whether an individual possessed mtDNA α or β clade haplotype, all individuals belonged to one nuclear gene lineage for the two X-linked (BGN and PHKA2) and one Y-linked (AMELY) genes sequenced. Analysis of multilocus genotypes indicated an average observed and expected heterozygosities were 0.543 and 0.539 in wild-sourced and 0.579 and 0.547 in the captive-born Asian elephants, respectively. No subdivision among the sampled individuals was detected, including data partitioned by mtDNA clades. Aside from parent–offspring dyads, no further relationships were detected among wild-sourced and captive-born Asian elephants (average relatedness value <0.000).