Sequence analysis of the hypervariable regions (HVRs) of mitochondrial DNA (mtDNA) are routinely performed in forensic casework, however, there are still issues to be resolved, such as the existence of multiple errors in published databases or the limitations of individual discrimination in certain populations. Here, we analyzed the coding region of mtDNA in detail by examining 36 haplogroup (HG)-defining single nucleotide polymorphisms (SNPs) using amplified product-length polymorphisms (APLP) method in conjunction with sequence analysis of HVR1 and HVR2 to establish a methodology for forensically reliable and practical mtDNA testing. The mtDNAs from 217 unrelated Japanese were examined and could be classified into 27 haplogroups. By combining the data of the coding region with those of HVRs, genetic diversity was slightly increased from 0.9817 to 0.9888 for HVR1/HG and from 0.9967 to 0.9970 for HVR1/HVR2/HG, as compared to the results of HVRs only. Moreover, in most cases, reliability of the HVR data could be confirmed by haplogroup motif analysis. Our mtDNA profiling method can provide reliable data in a time and cost-saving way due to the rapid and economical nature of APLP analysis.