Mitochondrial DNA (mtDNA) is maternally inherited without recombination events and has a high copy number, which makes mtDNA analysis feasible even when genomic DNA is sparse or degraded. Here, we present a SNP typing assay with 33 previously described mtDNA coding region SNPs for haplogroup assignment of Caucasians. Samples may be ascribed into eight major Caucasian haplogroups and 28 of the most frequent Caucasian subhaplogroups. The assay consists of a multiplex PCR reaction followed by two single base extension (SBE) reactions. The assay was tested on 49 unrelated Danish individuals previously typed by sequencing of the mitochondrial HV1 and HV2 regions. Haplogroup assignments based on mtDNA coding region SNPs and sequencing of HV1 and HV2 regions gave identical results for 27% of the samples, and except for one sample, differences in haplogroup assignments were at the subhaplogroup level.