The beta-subunit of human chorionic gonadotropin (hCG beta) is reportedly encoded by as many as seven non-allelic genes or pseudogenes. Previous studies have identified a cluster of three hCG beta gene copies and the single-copy lutropin-beta subunit (LH beta) gene, but overlap of this cluster with two additional pairs of hCG beta genes has not been demonstrated, despite the isolation of 18 genomic clones. To define the number and organization of non-allelic hCG beta gene copies, genomic Southern blot analyses were performed using hCG beta cDNA and gene-flanking unique-sequence probes. The data show the linear arrangement of six genes (or pseudogenes) and show that the hCG beta-IH beta gene cluster is present in a single 58-kilobase EcoRI fragment. Our map of the cluster indicates which of the cloned hCG beta genes reflect somatic genotypes rather than recombinant artifacts and will thus permit investigation of factors regulating expression during gestation.