Methylglyoxal (MG) acts as a toxic material in various species, known as a reactive α-oxoaldehyde having potential risk in disease. But it can be degraded by the in vivo biochemical pathway. Genes related to methylglyoxal were screened in Escherichia coli MG1655 strain. Among them, five unknown genes, yahA, yjhT, ydbD, yqiI, and yjjQ were chosen as candidates. Deletions of these genes showed lethality to methylglyoxal and dihydroxyacetone (DHA) in minimal media. When crude extracts of the deleted strains were assayed to detect methylglyoxal conversion activity by NMR, MG1655ΔgloΔyqiI exhibited reduced acetol and lactate productions. MG1655ΔgloΔydbD also showed lower level of lactate production than that of MG1655Δglo, suggesting that ydbD plays a role in the pathway to lactate production.