Natural ventilation is defined as the number of air exchanges per hour per unit floor area necessary
to reduce high indoor air temperature and humidity. In addition, it maintains the concentration of carbon dioxide. Natural ventilation is preferred in mechanical system as the ventilation opening is built into the greenhouse, with lower construction cost and no energy and maintenance inputs are required. A mathematical model to quantify natural ventilation rates was developed and verified in large-scale greenhouse structures. For this purpose, four Naturally Ventilated Tropical Greenhouse Structures were designed and constructed at the Malaysian Agricultural Research and Development Institute (MARDI). These were single, double, triple, and quadruple span structures with floor areas of 500 m2, 1000 m2, 1500 m2 and 2000 m2, respectively. This paper presents the validation of a mathematical model which was developed to quantify natural ventilation rates which are very crucial to reduce high in-house temperature built up in the tropics. Regression equations of natural ventilation against wind speed were found to be Φw = 0.0632V, Φw= 0.0395V, Φw= 0.0316Vand Φw=0.0276V for the single, double, triple and quadruple spans, respectively. Meanwhile, coefficients of determination showed strong relationships between ventilation rate and wind speed, with R2 = 0.9999 for all structures. Larger floor area was found to have higher in-house temperature than smaller ones. Ventilation rate inside the single-span structure was found to be higher compared to the multi-span structures, which increased linearly with the increasing wind speed at the eaves of structure.