Abstract: Pig-on-litter system is a new pig-raising technology that can reduce environmental pollution. In this system, micro-organisms are generally considered as the key factor. In order to determine the change in microbial community during fermentation and to set up basic data for scientific management of pig-on-litter system, microbial community of litter with different fermentation levels was analyzed using phospholipid fatty acid (PLFA) biomarkers. Fermentation levels of litters were divided into three grades (1^st, 2^nd and 3^rd) using the chromatic aberration (△E) method. Both surface (0-15 cm) and inner layer (30-45 cm) litters of each fermentation level were sampled. PLFA composition of each sample was determined by the Sherlock MIS 4.5 system. The results showed that the method used was sufficient to detect total 61 kinds of PLFA biomarkers. The most and least kinds of PLFA biomarkers occurred in litters with the 2^nd and 3^rd fermentation levels, respectively. PLFA biomarkers displayed the same order of distribution abundance in all samples-bacteria > fungi > actinomycetes. The contents of PLFAs that were referable to bacteria, fungi, actinomycetes, G⁺, G^- and total PLFA in the surface layer samples were all higher than those in the inner layer samples; being highest in surface layer litters with the 1^st fermentation level. Fermented litter had significantly higher content of total PLFA than unfermented litter (CK) (P < 0.05). The highest fungi/bacteria and G⁺/G^- ratios were for surface layer litter with the 3^rd and 2^nd fermentation levels, respectively. Diversity analyses indicated that the maximum values of the Shannon index and Pielou index were for litter of the 2^nd fermentation level and the maximum values of the Simpson index were for surface layer litter of the 3^rd fermentation level. Based on cluster analysis, the samples were clustered into three groups for Euclidean-distance of 223.15. Samples with the same fermentation level were clustered together. Also based on principal component analysis, surface layer and inner layer samples of the 1^st fermentation level were clustered into one lone group, while the other samples were clustered into other several groups. Put together, litters with different fermentation levels had different microbial community structures. The maximum values of microbial content and species were in surface layer litter with the 1^st and 2^nd fermentation levels, respectively. Moreover, surface layer and inner layer litters of the same fermentation level had similar microbial communities.