Objective Gas chromatography (GC) was used to investigate the cellular fatty acid (CFA) composition of 141 Acinetobacter baumannii and 32 A. calcoaceticus isolates from different locations in China and to find chemical markers to differentiate these two closely related bacteria. Methods Whole cell fatty acid methyl esters (FAMEs) were obtained by saponification, methylation, and extraction for GC analysis, followed by a standardized Microbial Identification System (MIS) analysis. Results All A. baumannii and A. calcoaceticus strains contained some major fatty acids, namely, 18:1 ω9c, 16:0, Sum In Feature 3, 12:0, 17:1ω8c, 3-OH-12:0, 17:0, Sum In Feature 2, 2-OH-12:0, and 18:0 compounds. Although most of the total CFAs are similar between A. baumannii and A. calcoaceticus strains, the ratios of two pairs of CFAs, i.e., Sum In Feature 3/18:1 ω9c versus 16:0/18:1 ω9c and Sum In Feature 3/18:1 ω9c versus unknown 12.484/18:1 ω9c fatty acids, could differentiate these two closely related bacteria. A. baumannii could be easily classified into two subgroups by plotting some ratios such as Sum In Feature 3/16:0 versus 17:0 and Sum In Feature 3/2-OH-12:0 versus17:0 fatty acids. Conclusion The ratios of some CFAs could be used as chemical markers to distinguish A. baumannii from A. calcoaceticus. Objective Gas chromatography (GC) was used to investigate the cellular fatty acid (CFA) composition of 141 Acinetobacter baumannii and 32 A. calcoaceticus isolates from different locations in China and to find chemical markers to differentiate these two closely related bacteria. Methods Whole cell fatty acid methyl esters (FAMEs) were obtained by saponification, methylation, and extraction for GC analysis, followed by a standardized Microbial Identification System (MIS) analysis. Results All A. baumannii and A. calcoaceticus strains contained some major fatty acids, namely, 18 : 1 ω9c, 16: 0, Sum In Feature 3, 12: 0, 17: 1ω8c, 3-OH-12: 0, 17: 0, Sum In Feature 2, compounds. Most most of the total CFAs are similar between A. baumannii and A. calcoaceticus strains, the ratios of two pairs of CFAs, ie, Sum In Feature 3/18: 1 ω9c versus 16: 0/18: 1 ω9c and Sum In Feature 3/18: 1 ω9c versus unknown 12.484 / 18: 1 ω9c fatty acids, could differentiate these two closely relat ed bacteria. A. baumannii could be easily classified into two subgroups by plotting some ratios such as Sum In Feature 3/16: 0 versus 17: 0 and Sum In Feature 3/2-OH-12: 0 versus 17: 0 fatty acids. Conclusion The ratios of some CFAs could be used as chemical markers to distinguish A. baumannii from A. calcoaceticus.