Mitochondrial DNA sequences from the 12S rRNA gene, four tRNA genes, and a portion of two protein coding genes were used to investigate the relationship of myliobatoid genera. In addition, we conducted an investigation of the sister group to the freshwater stingrays by sampling additional DNA sequences from GenBank. Consequently, two datasets were used to examine myliobatoid relationships. The first consisted of the genes sequenced in this study. The second dataset was compiled by combining the first dataset with cytochrome b sequences from GenBank. The second dataset, however, included a number of missing characters due to differences in sampling. The effect of the missing characters on both maximum parsimony and maximum likelihood analysis was investigated by conducting a simulation study. Results of the simulation study indicated that maximum likelihood was not sensitive to the missing data, whereas the accuracy of maximum parsimony analysis was expected to decrease. Phylogenetic analysis of this group had several areas concordant with morphological studies, however, the analysis also revealed two novel relationships. In addition, placement of two taxa (Gymnura and Himantura) were dependent both on the dataset and analytical method used.