Human DRB genes encode beta chains of the major histocompatibility complex (MHC) class II molecules. Although nine DRB loci have been mapped to the short arm of chromosome 6, an individual chromosome contains only one to five loci and is classified into one of five major haplotypes. To elucidate the origin of human DRB loci and haplotypes, intron 1 sequences approximately 5000 bp in length were determined for three DRB1 alleles (DRB1*03, DRB1*04, and DRB1*15) and five DRB genes (DRB2, DRB3, DRB4, DRB5, and DRB7). The sequences were subjected to phylogenetic analyses together with previously determined intron 4 and 5 sequences. The sequences provided two sources of information: Nucleotide substitutions that could be used to construct phylogenetic trees and to estimate divergence times and a set of insertions (mostly Alu elements) that reveal the order of splitting of duplicated genes. The combined data indicate that the ancestor of the human DRB genes was HLA-DRB1*04-like and that the DRB2, DRB7, DRB5, and DRB3 genes arose from this ancestor by four rounds of duplication 58, 56, 53, and 36 million years (MY) ago, respectively. The DRB4 gene may have arisen 46 MY ago by a deletion from the DRB1 and DRB2 genes and the DRB6 gene is probably an allele at the DRB2 locus. During the course of its evolution, the DRB1*04 gene acquired an intron 1 segment (including two Alu elements) from a gene that became the ancestor of DRB1*03. The present-day HLA-DR haplotypes were derived from three principal ancestral haplotypes: DRB1-DRB2, DRB1-DRB5, and DRB1-DRB7.