As assay for lipophilic pigments in phototrophic microbial mat communities using reverse phase-high performance liquid chromatography was developed which allows the separation of 15 carotenoids and chloropigments in a single 30 min program. Lipophilic pigments in a laminated mat from a commercial salina near Laguna Guerrero Negro, Baja California Sur, Mexico reflected their source organisms. Myxoxanthophyll, echinenone, canthaxanthin, and zeaxanthin were derived from cyanobacteria; chlorophyll c, and fucoxanthin from diatoms; chlorophyll a from cyanobacteria and diatoms; bacteriochlorophylls a and c, bacteriophaeophytin a, and gamma-carotene from Chloroflexus spp.; and beta-carotene from a variety of phototrophs. Sensitivity of detection was 0.6-6.1 ng for carotenoids and 1.7-12 ng for most chloropigments. This assay represents a significant improvement over previous analyses of lipophilic pigments in microbial mats and promises to have a wider application to other types of phototrophic communities.