We have generated a recombinant Mos1 transposon that can insert up to 45-kb transgenes into the Caenorhabditis elegans genome. The minimal Mos1 transposon (miniMos) is 550 bp long and inserts DNA into the genome at high frequency (~60% of injected animals). Genetic and antibiotic markers can be used for selection, and the transposon is active in C. elegans isolates and Caenorhabditis briggsae. We used the miniMos transposon to generate six universal Mos1-mediated single-copy insertion (mosSCI) landing sites that allow targeted transgene insertion with a single targeting vector into permissive expression sites on all autosomes. We also generated two collections of strains: a set of bright fluorescent insertions that are useful as dominant, genetic balancers and a set of lacO insertions to track genome position.