We demonstrate the applicability of ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry (ICEMS) for the simultaneous characterization of length and nucleotide polymorphisms. Two sections within the first (HVS-I) and second (HVS-II) hypervariable segments of the mitochondrial (mt)DNA control region were selected as targets, both containing poly-cytosine (C) tracts, which display length heteroplasmy at a substantial frequency in the population. The two mtDNA sections were simultaneously amplified and analyzed by ICEMS in 90 maternally unrelated mother-offspring pairs from Austria. The findings were confirmed by direct sequencing of the polymerase chain reaction products. For the detailed characterization of present-length heteroplasmic variants, the results retrieved through ICEMS were more informative compared with those derived from direct sequencing. Hence, ICEMS represents an interesting option for successful application in forensic science.