Pseudomonas aeruginosa is an important pathogenic and environmental bacterium, with the most widely studied strain being PAO1. Using the PAO1 reference cosmid library and the recently completed PAO1 genome sequence, we have mapped a minimal tiling path across the genome using a two-step strategy. First, we sequenced both ends of a set of over 500 random and previously mapped clones to create a backbone. Second, we end-sequenced a second set of cosmid clones that were identified to lie within the larger gaps using hybridization of the reference library filters with probes designed against sequences at the center of each gap. The minimal tiling path was calculated using the program Domino (http://www.bit.uq.edu.au/download/), with the overlap between adjacent clones set to 5 kb (where possible) to minimize the chance of truncating genes. This yielded a minimal tiling cosmid library (334 clones) covering 93.7% of the genome in 57 contigs. This library has reduced to a workable set the number of clones required to represent the majority of the P. aeruginosa genome and gives the precise location of each cosmid, enabling most genes of interest to be located on clones without further screening. This library should prove a useful resource to accelerate functional analysis of the P. aeruginosa genome.