Primary amenorrhea (PA) is a heterogenous condition with multifactorial etiologies and cannot be established with single diagnostic criterion. We have attempted to assess several factors and found significant association of body mass index, birth history, lifestyle factors like addiction habits, maternal history of proband; family history of consanguinity, amenorrhea and menstrual cycle; serum levels of follicle-stimulating hormone (FSH), luteinizing hormone (LH), thyroid-stimulating hormone (TSH), prolactin, estrogen, progesterone and testosterone with PA when compared with age-matched control females. Chromosomal abnormalities like Turner syndrome, presence of Y chromosome, Turner mosaics, Xp deletion, isochromosome X, and normal variants in PA females were observed. Chromosomal abnormalities were significantly associated with development of secondary sexual characters (SSCs) and ovaries in PA females. This study will help clinicians to understand the exact etiology and identify the best treatment option to improve of life of female patients and their families.