Positional cloning of an insect-resistance quantitative trait locus (QTL) requires the construction of a large-insert genomic DNA library from insect-resistant genotypes. To facilitate cloning of a major defoliating insect-resistance QTL on linkage group M of the soybean genetic map, a bacterial artificial chromosome (BAC) library for PI 229358 was constructed and characterized. The HindIII BAC library contains 55,296 clones with an average insert size 131 kb. This library represents a 6-fold soybean haploid genome equivalents, allowing a 99.8% probability of recovering any specific sequence of interest in soybean. BAC filters were screened with a genomic DNA probe Sat_258sc2 obtained through genome walking from flanking sequences of a simple sequence repeat (SSR) marker, Sat_258, which links to the insect-resistance QTL. Thirteen BAC clones were identified positive for Sat_258sc2, and two of them were confirmed to carry Sat_258. The results suggest that this library is useful in positional cloning of the major insect-resistance QTL, and the approach presented here can be used to screen a BAC library for a SSR marker without requiring the creation of BAC pools.