In the present study the role of the COL2A1 gene variable number of tandem repeats (VNTR)) polymorphism in the susceptibility to dental fluorosis in an endemically fluorosed district of Salem, Tamil Nadu, India, was investigated. Genomic DNA was extracted from the dental pulp of extracted teeth and the polymerase chain reaction (PCR) was done by the Qiagen protocol. The COL2A1 gene was sequenced using a 3730 DNA sequencer and the allelic ratio was identified by using gene marker software. Intergroup comparisons of alleles and genotypes with the presence and absence of dental fluorosis was found to be highly significant, OR=6.83, 95% CI=4.74-9.85, p-value<0.001, and OR=34, 95% CI=9.6-119.6, p-value<0.001, respectively. In the present study, we identified a total of 6 alleles and of 15 genotype combinations. The most frequent allele was 15R and the most frequent genotype was 12R/12R in the fluorosed group. In conclusion, a variable number of tandem repeats polymorphism of the COL2A1 gene was found to be strongly associated with dental fluorosis in the population studied.