Background: Amenorrhea is defined as the absence of menstruation in women at reproductive age, caused by imbalanced hormonal interactions. The genes located on X chromosome are linked to the physiology of menstruation and reproduction, and early detection of major chromosomal conditions can be facilitated through cytogenetic testing. This study aimed to determine the frequency and spectrum of chromosomal anomalies in amenorrhea patients from Eastern India and correlate clinical features with cytogenetic findings. Methods: From September 2022 to September 2024, 231 women with confirmed amenorrhea were included in a study conducted at inDNA Life Sciences Private Limited, India. Clinical features of women with a history of amenorrhea were recorded and cytogenetic investigation was carried out for all women. Results: Study revealed that 20.35% of amenorrhea cases exhibited chromosomal anomalies. Among them, 38.30% were classified as numerical anomalies, 25.53% as sex reversal, 19.15% as structural anomalies, and 17.02% as mosaic karyotypes, with X-monosomy identified as the most prevalent anomaly. Conclusion: The findings emphasizes the importance of karyotyping in diagnosis, highlighting its role in early detection and management of female infertility. Karyotyping has a resolution limit of 4-5 Mb, which disables identification of submicroscopic chromosomal abnormalities. In contrast, chromosomal microarray (CMA) analysis can examine the entire genome at higher resolutions, allowing for the identification of genetic abnormalities that may not be detected by karyotyping. While CMA was excluded from this investigation, it could serve as a valuable technique for future research aimed at identifying submicroscopic chromosomal abnormalities in cytogenetically normal women with amenorrhea.