Estrogens have critical roles in central regulation of reproduction. Their signaling is mediated mainly through activation of nuclear estrogen receptors. To date, two types of estrogen receptors have been documented: estrogen receptor α (ERα, which is known as ESR1) and estrogen receptor β (ERβ, as ESR2). The estrogen receptor genes have multiple promoter systems, and the transcribed pre-mRNAs are subject to complex splicing. However, selective promoter usage and splicing patterns of the 5’-untranslated region (5’-UTR) variants of the human ERβ gene have not been fully characterized. Therefore, we examined the presence of human ERβ variants with unique 5’-UTRs using rapid amplification of cDNA 5’-ends and RT-PCR, and determined the genomic organization of the human ERβ gene. Furthermore, we analyzed alternative promoter usage and alternative splicing profiles of the human ERβ gene in a wide range of peripheral organs and brain subregions. The genomic analysis revealed that the gene contained three promoters (0K, 0N, and E1 promoters) and promoter-specific leader exons, and several untranslated internal exons in the 5’-region. Alternative inclusion of the internal exons between exons 0K and 1 yielded multiple splice variants in the testis and brain subregions. The promoter-specific isoforms exhibited distinct expression patterns. In particular, the human-specific 0K isoforms were strongly expressed in the testis and widely distributed in the brain. These findings provide fundamental and useful information for further analyses on the regulatory mechanisms of human ERβ expression.