One of the most important features of urothelial cancers of the bladder and upper urinary tract is metachronous and/or synchronous multifocal occurrence with high frequency. Since such multifocal recurrent tumors are derived from a common transformed cell, the chronological tracing of genetic alterations in such multifocal tumors may reveal the precise timing and role of genetic alterations in urothelial carcinogenesis. In this study, we tested the presence of microsatellite alterations in synchronous and/or metachronous multifocal urothelial cancers to examine the chronological genetic alterations for the presence of hierarchy of genetic alterations in urothelial cancer development. Genetic alterations at 20 microsatellite loci on 8 chromosomal arms (2q, 4p, 4q, 8p, 9p, 9q, 11p, and 17p) were tested. Judging from the patterns of allelic deletion and microsatellite shifts, multifocal tumors in at least 21 (81%) of the 26 evaluable patients were considered to be derived from a single progenitor cell. In patients with multifocal tumors of an identical clonal origin, discordant microsatellite alterations were observed at significantly lower frequencies on chromosome 9 compared with those on the other chromosomes tested. The heterotopic spread and genetic divergence may occur long before the clinical manifestation of multiplicity from a single transformed cell. The data strengthens the previous view that heterotopic spread of transformed progenitor cells and genetic divergence occur after chromosome 9 alterations in most of urothelial cancers.