Introduction: Male infertility is a serious problem in developing world where genetic and epigenetic factors play a crucial role in the pathogenesis of the disease. The rationale behind the present study to understand the genetic basis of male infertility, to identify the “novel gene mutation” and also asses the frequency (%) of microdeletion of Y-chromosome i.e. deletion of AZF regions interfare during spermatogenesis. Still, 10-20% cases of infertility fail to identify exact cause of male infertility and are fall in the category of unexplained cause of infertility in non obstructive azoospermia. Material and Methods: Blood samples were collected from the cases of clinically diagnosed non obstructive azoospermia (NOA) with respective controls. Study was performed using RT-PCR based analysis using 14 set of STS markers of AZF region allocated on Y- chromosome and NextGen Sequencing. Results: Mutational spectra include the individual variations of frequency of AZF gene mutation as a factor responsible for male infertility in eastern part of the country. Genetics analysis of AZF a, b, and c regions showing different frequency of deletion but the deletion of AZFc showing significant difference with respect to controls (p<0.001). NGS play a significant role to explore the involvement of de novo mutation of USP9Y and PCDH11Y gene mutation resulting changes in protamines. The deletion frequency AZFa region is 1.0%, while AZFb and AZFc regions showing 6% & 19%, respectively in non obstetric azoospermic cases. Hence, curiosity has been developed further to identify “new mutations” based on Next Gen Sequencing, identifies USP9Y gene of AZFa region showing non-frame shift mutation (insertion of C→G/C→A) at region exon42:c.6996_6997 insCGA in heterozygous condition. Secondly, of AZFb region showing single nucleotide gene polymorphism rs2524543, G→T and rs2563389, T→ G of PCDH11Y gene in homozygous condition. Conclusion: The identification of causative mutations in the cases of NOA and their penetrance lead to interference in spermiogenesis .Hence, on the basis of mutational spectra, genetic counselling of infertile couples are required before reaching to final decision. No doubt the environmental factors influence the gene-pool lead to altered spermatogenesis in male infertility.