The organization of genomic sequences is dynamic and undergoes change during the process of evolution. Many of the variations arise spontaneously and the observed genomic changes can either be distributed uniformly throughout the genome or be preferentially localized to some regions (hot spots) compared to others. Conversely cold spots may tend to accumulate very few variations or none at all. In order to identify such regions statistically, we have developed a method based on Shewhart Control Chart. The method was used for identification of hot and cold spots of single-nucleotide variations (SNVs) in Mycobacterium tuberculosis genomes. The predictions have been validated by sequencing some of these regions derived from clinical isolates. This method can be used for analysis of other genome sequences particularly infectious microbes.