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Since the 1980s, chromosome-integration vectors have been used as a core method of engineeringBacillus subtilis. One of the most frequently used vector backbones contains chromosomally derived regions that direct homologous recombination into theamyElocus. Here, we report a gap in the homology region inherited from the originalamyEintegration vector, leading to erroneous recombination in a subset of transformants and a loss-of-function mutation in the downstream gene. Internal to the homology arm that spans the 3′ portion ofamyEand the downstream geneldh, an unintentional 227 bp deletion generates two crossover events. The major event yields the intended genotype, but the minor event, occurring in ~10 % of colonies, results in a truncation ofldh, which encodes lactate dehydrogenase. Although both types of colonies test positive foramyEdisruption by starch plating, the potential defect in fermentative metabolism may be left undetected and confound the results of subsequent experiments.