Background: Male infertility is a multifactorial medical problem affecting around 7% of the male population in whom 1% are affected by Non Obstructive Azoospermia (NOA). Despite of advanced diagnostic techniques, majority of the infertility cases remain idiopathic .Therefore more evidence based scientific research is required to investigate the underlying cause of this medical problem. The objective of the current study was to investigate the etiology of idiopathic primary infertility in an extended consanguineous infertile family using clinical and molecular cytogenetic diagnostic techniques. Material and methods: A comprehensive questionnaire was filled from the available members and blood was sampled from 3 infertile patients and 2 controls brothers along with parents of a consanguineous infertile family. Semen analyses, Reproductive hormones, Multiplex PCR, cytogenetic and molecular analyses were performed. Moreover, testicular biopsy from one patient was collected and subsequently hematoxylin and eosin staining along with meiotic surface spreading was performed on testicular section. H. Muhammad, et al. Genetics and Molecular Research 19 (5): gmr16039992 Finally, we did Whole Exome Sequencing (WES) to identify any genetic variants in the idiopathic infertile subjects and fertile controls. Results: Among the 4 infertile male individuals, testicular histological analysis of one patient (IV:8) revealed spermatocyte arrest. Subsequently, abnormal pachytene was observed and the overall population of normal pachytene was significantly reduced in patient as compared to control. WES approach was used to find out the possible genetic cause of idiopathic primary infertility and we have identified a novel homozygous missense recessive mutation in ZNF621 (exon3:c.82C>G, p.L28V) that was co-segregating with infertility phenotype. Identified mutation was predicted deleterious by various softwares which might be associated with spermatogenic failure and primary infertility. Discussion: In summary, all the studied subjects had normal 46XY karyotype and no AZF microdeletion was found .We detected a novel genetic variant in an unknown gene ZNF621 which might be associated with disrupted spermatogenesis and infertility. Further research on large scale with recent evidences are required to diagnose the underlying cause of idiopathic primary male infertility and link the unknown role of ZNF621 in spermatogenesis and normal progression of meiosis. Conclusion: Despite extensive laboratory investigations, millions of infertile men are affected by non-obstructive azoospermia, in whom many of them are diagnosed as idiopathic. A sound proportion of these affected individuals may be investigated by WES at the point of care. While screening this family, we have found a novel missense recessive mutation in an unknown ZNF621 gene by screening an extended consanguineous infertile Pakistani family. Further large scale research is recommended to explore the unknown role of ZNF621 and its association with idiopathic primary infertility.