The DNA genomes of human T-lymphotropic virus type III (HTLV-III) isolated from 18 individuals with AIDS or who were at risk for AIDS were evaluated for evidence of variation. Although all of the 18 viral DNA's hybridized throughout their entire genomes to a full-length cloned probe of the original HTLV-III isolate, each of the 18 isolates showed a different restriction enzyme pattern. The number of restriction site differences between isolates ranged from only 1 site in 23 to at least 16 sites in 31. No particular viral genotype was associated with a particular disease state and 2 of the 18 patients had evidence of concurrent infection by more than one viral genotype. Propagation of three different viral isolates in vitro for up to 9 months did not lead to detectable changes in their restriction patterns. These findings indicate that different isolates of HTLV-III comprise a spectrum of highly related but distinguishable viruses and have important implications regarding the pathogenicity of HTLV-III and attempts to develop effective diagnostic, therapeutic, and preventive measures for this virus.