Three general recommendations are proposed when insertions and deletions (gaps) in human mtDNA vary, which may cause laboratories to code mtDNA differently: (1) profiles should be characterized in such a way that the least number of differences from the reference sequence are present; (2) differences should be prioritized according to insertions/deletions, transitions, and transversions; and (3) insertions and deletions should be placed 3 inches with respect to the light strand of human mtDNA and should be combined in situations where the same number of differences from the references sequences is maintained. Several examples are presented in which alternative alignment strategies resulted in different characterizations of mtDNA profiles. All alignment examples were drawn from an expanded version of the Scientific Working Group on DNA Analysis Methods. These recommendations and examples are presented in an effort to standardize the treatment of length variation in human mtDNA within the forensic community. The authors maintain that the current method of recording differences from a reference is preferred and should be continued in order to facilitate communication. The authors also stress the importance of continued discussion of the proposed rules.