At 600 kb, the genome of Mycoplasma genitalium is among the smallest known for cellular organisms capable of independent replication. As such, elucidation of the genetic makeup and chromosome architecture of this organism is of considerable interest. We have located 631 markers on the physical map of M. genitalium. The clones have been mapped by hybridizing 20 overlapping cosmid and lambda clones which encompass the entire M. genitalium chromosome to replica filters containing 856 genomic DNA clones. Three hundred fifty-six of these clones represent sequence tag sites, which were previously characterized by database searches. The remaining markers represent clones with an average size of 2.5 kb derived from Sau3A1 partial digestion of genomic DNA. The hybridization data can be divided into three classes: clones which hybridized to only one cosmid; clones which hybridized to two adjacent and overlapping cosmids; and clones which hybridized to several cosmids, which represent repetitive DNA. This rapid approach for placing clones on the physical map has allowed useful comparisons to be made with other bacterial chromosomes, especially that of the closely related organism M. pneumoniae, and has provided insight to the types of events which may have led to the reduction in size of this genome. Future use of these data is discussed.