Large Insert Genome Analysis (LIGAN) is a broadly applicable, high-throughput technology designed to characterize genome-scale structural variation. Fosmid paired-end sequences and DNA fingerprints from a query genome are compared to a reference sequence using the Genomic Variation Analysis (GenVal) suite of software tools in order to pinpoint locations of insertions, deletions and rearrangements. Fosmids spanning regions that contain new structural variants can then be sequenced. Clonal pairs of Pseudomonas aeruginosa isolates from four cystic fibrosis patients were used to validate LIGAN technology. Approximately 1.5 Mb of inserted sequences were identified, including 742,869 bp containing 651 ORFs that are absent from published P. aeruginosa genomes. Six rearrangement break points and 220 kb of deleted sequences were also identified. Our study expands the “genome universe” of P. aeruginosa and validates a technology that complements emerging, short-read sequencing methods that are better suited to characterizing single-nucleotide polymorphism than structural variation.