We aimed to determine the genetic diversity, phylogenetic relationship and transmission dynamics of Mycobacterium tuberculosis complex (MTBC) genotypes in an area with high tuberculosis (TB) incidence. A set of 164 MTBC isolates from new TB patients of Golestan province, Iran, were subjected to genotyping using the standard 24-locus MIRU-VNTR method. Recent TB transmission was evaluated and phylogenetic relationships were analysed by minimum spanning tree and cluster-graph methods. Among the 164 isolates, 132 distinct patterns were detected. The 48 clustered isolates (29.3%) were distributed into 16 clusters ranging in size from 2 to 12 isolates. The most frequent genotype was Central Asian Strain/Delhi (CAS/Delhi) (n = 67, 40.8%), followed by NEW-1 (n = 53, 32.3%) and Beijing (n = 19, 11.6%) genotypes. Thirty five (72.9%) of NEW-1 isolates were recovered from immigrant patients and 84.2% (n = 16) of Beijing genotypes recovered from native cases. Statistically significant association was found between clustering and smoking (p = 0.047), drug addiction (p = 0.01) and prison history (p = 0.003). The estimated proportion of recent transmission was 19.5%. Presence of highly diverse MTBC isolates circulating in this province without a dominant genotype might be a consequence of importation of various genotypes in this area.