Psychrophiles and Psychrotolerants are important resources of biologically active substances and cold-adapted enzymes. A total of 33 cold-adapted strains, including 6 Gram-positive strains and 27 Gram-negative strains, were isolated from soil samples. The analysis of these membrane fatty acids showed that the branched chain fatty acids were the major lipid pattern in Gram-positive bacteria, which may indicates their main cold adapted mechanism. However, the membrane fatty acids of Gram-negative bacteria were various, including unsaturated, branched and short-chain fatty acid. Species identifications were committed for 17 strains with typical fatty acid compositions. They were distributed in 11 genus. Variation of cell membrane lipid composition was consistent with the result of 16S rRNA. In addition, the Gram-negative bacteria exhibited the different strategies on cold adaptation, even that these strains mainly contained the unsaturated fatty acid. Related researches would not only represent cold adapted mechanism of cold-adapted microorganisms, but also provide the precious resources for the utilization of cold-adapted enzymes.