A survey method is presented for the unbiased sampling and identification of bacterial species. The method utilizes a randomized selection process and the MIDI Microbial Identification System (MIS) which uses whole cell fatty acid analysis by gas chromatography rather than relying on colonial growth morphology and conventional biochemical testing. Approximately 1093 bacterial isolates were made and identified from cotton. The method uncovers a greater diversity of bacterial species from cotton than has hitherto been reported. In California, the bulk of bacterial species consisted of Bacillus spp.; and in Mississippi and Texas region cottons, the bulk of bacterial species consisted of Pseudomonas spp. No significant differences between populations were observed in the nonsticky, moderately sticky and sticky cottons. A Gram-index concept is introduced which relates the ‘Gram-reaction character’ of a cotton growing region.