We investigated polymorphic Alu insertion (POALIN) frequencies at five loci in the major histocompatibility complex (MHC) class I genomic region to determine their allele and haplotype frequencies and associations with the human leukocyte antigen (HLA)-B and -Cw genes in seven different Chinese ethnic populations, the Han, Bulang, Wa, Dai, Maonan, Hani and Jinuo. The POALINs varied in frequency between 0% and 42.3% with significant differences between populations at all of the loci. Each POALIN was in significant linkage disequilibrium with a variety of HLA-B or -Cw four-digit alleles. The percentage association between Alu insertions and the HLA-B or -Cw alleles was calculated in pairwise analyses of haplotypes to show possible crossing over events between loci. The POALIN insertions also helped to further stratify the HLA-B:-Cw haplotypes into different POALIN:HLA-B:HLA-Cw haplotype frequencies. Of the two-locus, five-locus and seven-locus haplotype analyses, the seven-locus haplotypes showed the largest number of differences between the populations. The most common multilocus haplotype in Han was MICB*1:B*4601:Cw*0102:TF*1:HJ*1:HG*2:HF*1 (15.6%) associated with the AluHG insertion, whereas the second most common multilocus haplotype in Han was MICB*1:B*1502:Cw*0801:TF*1:HJ*2:HG*1:HF*1 (11.8%) associated with the AluHJ insertion. This comparative study of multilocus POALINs in the HLA class I region of seven Chinese ethnic populations shows that POALINs alone or together with the HLA class I alleles are informative genetic markers for the identification of HLA class I allele and haplotype lineages and variations such as crossing over events within the same and/or different populations.