There are marked differences between wet and freeze-dried cells with regard to the identification of polar lipid components. The determination of the polar lipid composition of freeze-dried cells is well established. However, several approaches to identifying polar lipid components in wet cells have met with limited success owing to the presence of non-polar compounds in the extracts, resulting in a lipid composition with a narrow scope. In this study, we surveyed the lipid profiles of the wet biomasses of three Gram-positive (Microbacterium lacticum, Rhodococcus koreensis, and Streptomyces longwoodensis) and two Gram-negative (Pseudomonas aeruginosa and Novosphingobium capsulatum) bacteria; the results were comparable in quality to those obtained using a standard freeze-dried approach. Moreover, our improved method ensures simple lipid extraction. Overall, the results of the analysis showed minor lipid profile differences between the two approaches with regard to quantity, and lipid identification was consistent in both methods for all species.