Differently colored layers of freshwater hot spring mats at Boekleung (Western Thailand) were studied. Temperatures ranged from over 50 up to 57°C. Two mats were characterized: a laminated mat with a green and a red layers, and a monolayer, greenish-yellow mat. Bacterial communities in green, red, and yellow layers were investigated using molecular, culturing and pigment analysis methods. Pigment profiles covered a wide spectrum from chlorophylls to carotenoids. A green mat layer showed higher relative content of chlorophyll than yellow and red layers which presented higher proportion of carotenoids. Cyanobacterial isolates grow up to 55–56°C and their pigment profiles showed a relatively high content of chlorophylls suggesting the importance of other bacterial groups in the mat pigment profiles. Bacterial communities were analyzed by 16S rDNA surveys showing Cyanobacteria and Chloroflexi as the mayor components of the community. Other significant members were Candidate Division OP10, Bacteroidetes, Planctomycetes and Actinobacteria. These results highlight a major participation of Cyanobacteria and Chloroflexi in thermal mat communities, and the preferential presence of Candidate Division OP10 in green mat layers. Differently colored mat layers showed characteristic bacterial communities which could be discriminated from pigment profiles and molecular surveys.