We report the sequence of the Halobacterium salinarum strain R1 chromosome and its four megaplasmids. Our set of protein-coding genes is supported by extensive proteomic and sequence homology data. The structures of the plasmids, which show three large-scale duplications (adding up to 100 kb), were unequivocally confirmed by cosmid analysis. The chromosome of strain R1 is completely colinear and virtually identical to that of strain NRC-1. Correlation of the plasmid sequences revealed 210 kb of sequence that occurs only in strain R1. The remaining 350 kb shows virtual sequence identity in the two strains. Nevertheless, the number and overall structure of the plasmids are largely incompatible. Also, 20% of the protein sequences differ despite the near identity at the DNA sequence level. Finally, we report genome-wide mobility data for insertion sequences from which we conclude that strains R1 and NRC-1 originate from the same natural isolate. This exemplifies evolution in the laboratory.