The quality of ripe mango fruits depends on maturity stage at harvest, which is usually assessed by visible criteria or from estimates of the age of fruit. The present study deals with the potential of chlorophyll fluorescence as a nondestructive method to assess the degree of fruit maturity regardless of fruit growing conditions. Chlorophyll fluorescence parameters were measured along with respiration rates of fruits still attached to the tree. At the same harvest stage, based on the fruit age or the thermal time sum (degree-days) method, physical and biochemical measurements related to fruit maturity and quality were made. Shaded fruits had a significantly greener flesh color, as well as a lower fruit density and flesh dry matter content, than well-exposed fruits, showing that fruits at the top of the canopy were more mature than fruits within the canopy, which were still in a growth phase. Additionally, chlorophyll fluorescence parameters, Fo, Fm, and Fv, were significantly lower for fruits taken from the top of the canopy than for those from within the canopy. The unique relationship observed between chlorophyll fluorescence parameters and fruit maturity, estimated by internal carbon dioxide content, on fruit still attached to trees is independent of growing conditions, such as the position of the fruit in the canopy and carbohydrate supply. The chlorophyll fluorescence method evaluates maturity much more accurately than the degree-day method and, moreover, nondestructively provides values for individual fruits before harvest.