This study investigated the estimation of grain yields of three major annual crops in Ontario (corn, soybean, and winter wheat) using MODIS reflectance data extracted with a general cropland mask and crop-specific masks. Time-series two-band enhanced vegetation index (EVI2) was derived from the 8 day composite 250 m MODIS reflectance data from 2003 to 2016. Using a general cropland mask, the strongest positive linear correlation between crop yields and EVI2 was observed at the end of July to early August, whereas a negative correlation was observed in spring. Using crop-specific masks, the time of the strongest positive linear correlation for winter wheat was found between mid-May and early June, corresponding to peak growth stages of the crop. EVI2 derived at peak growth stages of a crop provided good predictive capability for grain yield estimation, with considerable inter-annual variation. A multiple linear regression model was established for county-level yield estimation using EVI2 at peak growth stages and the year as independent variables. The model accounted for the spatiotemporal variability of grain yields of about 30% and 47% for winter wheat, 63% and 65% for corn, and 59% and 64% for soybean using the general cropland mask and crop-specific masks, respectively. A negative correlation during the spring indicated that vegetation index extracted using a general cropland mask should be used with caution in regions with mixed crops, as factors other than the growth conditions of the targeted crops may also be captured by remote sensing data.