An efficient approach for structural studies on bacterial chromosomes is presented. It is based on high-resolution PCR map construction by using a multiplex long accurate PCR (MLA PCR) protocol and a YAC clone carrying the region to be mapped as indicator. The high-resolution PCR map of the Bacillus subtilis rrnB-dnaB region is presented as an example. Data are also presented on the use of DNA generated by LA PCR for sequencing; they are relevant to LA PCR induced mutations and justify the application of such mapping for sequencing long stretches of bacterial chromosomes.