In this study, the 5′ promoter region of MHC class I chain-related gene B (MICB) was investigated in 104 healthy, unrelated Han individuals recruited from northern China, using polymerase chain reaction (PCR)-sequencing methodology. Fifteen variable sites were detected, which showed a mosaic pattern of significant global linkage disequilibrium (LD). Eleven different 5′ promoter haplotypes were identified. MICB 5′ promoter haplotype-9 carrying CT deletion at positions −139/−138, which is associated with decreased MICB promoter activity, was present in 26.9% of MICB alleles and was linked to MICB*002:01, MICB*008, and MICB*014 in this population. In addition, rs3828913 (position −176) and rs3828914 (position −152) were located adjacent to HSRE and GC box in MICB promoter, respectively. Sixteen extended haplotypes (EH) incorporating the MICB 5′ promoter and MICB coding region were observed in this population, eight of which were in significant LD. Phylogenetic analysis of 5′ promoter refined MICB sub-lineage structure indicated that MICB*005:02, the most common MICB allele, consists of five sub-lineages. Ewens–Watterson homozygosity statistics at MICB 5′ promoter region were consistent with neutral expectations. Our study has shed new insight into MICB genetic variation at population level, suggesting that binding sites for transcription factors in MICB promoter could be interrupted by polymorphisms within this region, resulting in allele-specific regulation. The data will facilitate the understanding of regulation of MICB gene transcription, and will inform studies of evolution of the major histocompatibility complex (MHC) gene complex.