Technical Abstract: The ability to monitor mitochondrial background in Verticillium dahliae may provide an additional tool for population studies and monitoring clonal populations. Published mitochondrial genome sequences of V. dahliae (DQ531941) were used to design primers for assessment of mitochondrial haplotype differences among isolates. Five regions were examined (5,229 bp, or 19% of the total genome size) for 30 isolates representing a range in VCG, host, and geographic origin. Observed differences among isolates were due to single nucleotide polymorphisms, different numbers of bases in specific homopolymeric regions, and copies of subrepeated sequences. When all the differences observed for each locus was totaled there were polymorphisms at 28 loci; when the results of each locus for individual isolates were combined there were 15 mitochondrial haplotypes. Some of the observed grouping correlated with VCG. For example, five VCG-1 isolates from California, Spain and Greece had identical haplotypes, however, this was not observed for VCG-2 or VCG-4 isolates. While many VCG-2 isolates also were identical and fell into a single haplotype, polymorphisms were observed among some isolates and 7 haplotypes were found for this group (some of this corresponding to subgroupings –2A and –2B). Likewise, five VCG-4 isolates fell into 4 mitochondrial haplotypes, one of which was identical to the largest VCG-2 grouping. Phylogenetic analysis with these five regions revealed the mitochondrial background of VCG-1 to be monophyletic but VCG-2 and VCG-4 were polyphyletic. The results obtained indicate that there is variation in mitochondrial haplotypes and this type of analysis may be a useful for characterization of isolates.