Background: Methotrexate (MTX) was one of the first drugs synthesized for a specific chemotherapeutic purpose used to inhibit folic acid (FA) for the treatment of acute lymphoblastic leukemia in children. Its history is closely related to the discovery and characterization of folic acid. It is used clinically in medicine to treat a range of cancerous and noncancerous conditions. MTX is currently used in gynecology to treat disorders arising from trophoblastic tissue, namely, ectopic pregnancy. MTX, the most frequently used diseasemodifying anti-rheumatic drug (DMARD), suppresses disease activity and reduces joint damage. The aim of study: It is designed to demonstrate the effect of MTX (7.5 mg/wk.) on the histogenesis of gonad (testis) of newborn Albino rat. Material & Methods: Twenty pregnant female rats taken, classified equally to 10 control and 10 treated with MTX intramuscularly (first dose at day 15 of gestation and second dose at two day after birth). After normal vaginal delivery, newborns harvested at day seven. For histological study the newborn gonad (testis) were fixed in Bouin’s fixative, paraffin infiltration, and then embedded sections stained with haematoxylin and eosin, the specimens independently read. The result: The histological investigation showing atrophy of seminiferous tubules, increase interstitial space with reduce interstitial connective tissue and leydig cell, destruction of basement membrane, complete detachment of spermatogenic cell from basement membrane, atrophy of spermatogenic cell, swelling spermatogenic cell, degeneration sertoli and leydig cell, cell death, congested blood vessels and hemorrhage. The statistical analysis at day seven shown significant decrease (P<0.05) in newborn weight compared with control group. Highly significant increase (P<0.001) in diameter of testes. Significant decrease (P<0.01) in diameter of seminiferous tubules. Significant decrease (P<0.05) in numbers of spermatogenic cell in seminiferous tubules. The conclusion: The findings of the present study show that therapeutic dose of methotrexate is capable of producing histological changes in newborn’s testis at day seven