Primary amenorrhoea, defined as the absence of menstruation by age 14 without secondary sexual characteristics or by age 16 with them, is a clinical symptom with diverse etiologies. Objective: To determine the frequency and types of chromosomal abnormalities in patients presenting with primary amenorrhoea and to evaluate the clinical significance of cytogenetic analysis in diagnosis and management. Methods: A 25 cases detailed assessment including clinical history, physical examination, hormonal profiling, ultrasonography, and chromosomal analysis was conducted on individuals presenting with primary amenorrhoea, in human genetic lab from B.J. MEDICAL COLLEGE, AHMEDABAD, GUJARAT. Results: Chromosomal abnormalities were identified in a significant proportion of cases, ranging between 15.9% and 63.3%, with Turner’s syndrome, gonadal dysgenesis, and androgen insensitivity syndrome being the most frequently observed. Most anomalies involved numerical or structural abnormalities of the X chromosome. Conclusion: Cytogenetic evaluation is crucial in the diagnostic workup of primary amenorrhoea, aiding in accurate diagnosis, prognosis estimation, and guiding genetic counseling. Incorporating karyotyping into routine assessment enhances clinical decision-making and patient outcomes