A comprehensive study of the annual ovarian cycle in the house gecko, Hemidactylus flaviviridis, was conducted in Oman. Circulating estradiol (E2), progesterone (P), and testosterone (T) were measured during the active and inactive phases of the cycle. The steroid levels were related to the steroidogenic ultrastructural features such as the abundance of smooth endoplasmic reticulum (SER), the presence of cisternal whorls, and close association of lipid droplets with swollen vesiculated mitochondria and SER. The steroids were measured using a sensitive detection technique HPLC/MS/MS. E2 levels began to rise in January at the onset of vitellogenesis and continued to rise between February and May relative to ovulation,postovulation, gravidity and oviposition. Afterwards, E2 remained low during the inactive phase (June–December). P levels increased significantly in March and peaked in April, which coincided with luteunization. P levels began to decline relative to luteolysis (May–June). Afterwards, it remained low throughout the inactive phase. T levels rose significantly in March–April coinciding with vitellogenesis, but decreased rapidly and significantly in May and remained low during the inactive phase. Progesterone receptors (PR), identified using immunohistochemistry, were strongly expressed during the breeding period, but were absent during the non-breeding period. The appearance of the steroidogenic ultrastructural features in the preovulatory and lutein granulosa cells was correlated with the significant rise in the three steroid levels and the PR. As the steroid levels declined, the granulosa cells underwent a general degeneration and disruption of the associated steroidogenic features.