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Chlorophyll (Chl) content is among the most important crop biophysical characteristics. Chlorophyll can be related to photosynthetic capacity, thus, productivity, developmental stage, and canopy stresses. The objective of this study was to quantify and characterize the temporal variation of Chl content in the vertical profile of maize (Zea mays L.) canopies by means of a reflectance-based, nondestructive methodology. A recently developed technique that relates leaf reflectance with leaf pigment content has been used for accurate leaf Chl estimation. The technique employs reflectance in two spectral bands: in the red edge (720-730 nm) and in the near infrared (770-800 nm). More than 2,000 maize leaves were measured for reflectance and total and green area during a growing season. A bell-shaped curve showed a very good fit for the vertical distribution of Chl content regardless of crop growth stage. The parameters and coefficients of the bell-shape function were found to be very useful to interpret temporal changes in the vertical profile of each variable. Comparisons among Chl, leaf area index (LAI) and green LAI showed that Chl content was more sensitive to changes in the physiological status of maize than other biophysical characteristics. The quantification of Chl content in canopy should be seen as a useful tool to complement the information on green LAI or LAI. Its applicability will help to improve the understanding of the crop ecophysiology, productivity, the radiation use efficiency and the interplant competition.