Our variant ascertainment algorithm, VAAL, uses massively parallel DNA sequence data to identify differences between bacterial genomes with high sensitivity and specificity. VAAL detected approximately 98% of differences (including large insertion-deletions) between pairs of strains from three species while calling no false positives. VAAL also pinpointed a single mutation between Vibrio cholerae genomes, identifying an antibiotic's site of action by identifying sequence differences between drug-sensitive strains and drug-resistant derivatives.