Aims: To investigate the inter-strain variation in (i) substrate utilization and (ii) the restriction fragment length polymorphism (RFLP) pattern based on the distribution of an insertion element (IS1550) in Mycoplasma fermentans strains, and to establish any correlation between subgroups within the species and their source or habitat. Methods and Results: Using a sensitive dynamic pH method, the pattern and kinetics of substrate utilization by a panel of 17 M. fermentans strains from various sources was determined. This study correlated the biochemical characteristics of these strains with RFLP patterns based on the distribution of an insertion sequence (IS1550) with the sources of the strains. The test isolates were divided into four major groups according to the pattern of substrates metabolized. Interestingly, two strains isolated from cell lines in RFLP cluster I failed to utilize arginine. Ovine strains showed distinct substrate utilization patterns and produced RFLP patterns not previously encountered. Conclusions: All strains utilized glucose, but the ability to utilize arginine, fructose and N-acetyl glucosamine varied. There was also some correlation evident between the metabolic data and the RFLP clusters. Significance and Impact of the Study: This study has provided a better understanding of the biochemical and genetic diversity of M. fermentans strains from various sources.