In this study, we investigated the major histocompatibility complex (MHC) class I chain-related gene B (MICB) allelic variation by sequence-based typing (SBT) in 201 healthy, unrelated Han subjects from Hunan province, southern China. Eleven MICB alleles were observed, among which MICB*005:02 predominated with a frequency of 64.93%. Significant linkage disequilibrium (LD) was observed for 5 HLA-B-MICB and 6 MICA-MICB haplotypes. Compared with a northern Chinese Han population, several MICB-containing haplotypes appeared to be highly specific to this southern Chinese Han population. Two new MICB alleles, MICB*005:06 and MICB*026, were identified. Aligned with MICB*005:02, MICB*005:06 has a synonymous T replacement at nucleotide 762 in exon 4; MICB*026 has probably arisen from MICB*004:01 through a single nucleotide substitution from G to A at position 826 in exon 4, leading to an amino acid change from glutamic acid to lysine at codon 253. HLA-A*02-C*01-B*46-MICA*010-MICB*005:02-DRB1*09 was the most prevalent six-locus haplotype with a frequency of 8.49%. HLA-A*30-C*06-B*13:02-MICA*008:01-MICB*005:02-DRB1*07 appeared to be a conserved extended haplotype. Our results provide new information about MICB genetic polymorphism in Chinese Han populations, and will inform future studies of the potential role of MICB in allogeneic organ transplantation and disease susceptibility in related ethnic groups.