Research on fusion modeling of high spatial and temporal resolution images typically uses MODIS products at 500 m and 250 m resolution with Landsat images at 30 m, but the effect on results of the date of reference images and the 'mixed pixels' nature of moderate-resolution imaging spectroradiometer (MODIS) images are not often considered. In this study, we evaluated those effects using the flexible spatiotemporal data fusion model (FSDAF) to generate fusion images with both high spatial resolution and frequent coverage over three cotton field plots in the San Joaquin Valley of California, USA. Landsat images of different dates (day-of-year (DOY) 174, 206, and 254, representing early, middle, and end stages of the growing season, respectively) were used as reference images in fusion with two MODIS products (MOD09GA and MOD13Q1) to produce new time-series fusion images with improved temporal sampling over that provided by Landsat alone. The impact on the accuracy of yield estimation of the different Landsat reference dates, as well as the degree of mixing of the two MODIS products, were evaluated. A mixed degree index (MDI) was constructed to evaluate the accuracy and time-series fusion results of the different cotton plots, after which the different yield estimation models were compared. The results show the following: (1) there is a strong correlation (above 0.6) between cotton yield and both the Normalized Difference Vegetation Index (NDVI) from Landsat (NDVIL30) and NDVI from the fusion of Landsat with MOD13Q1 (NDVIF250). (2) Use of a mid-season Landsat image as reference for the fusion of MODIS imagery provides a better yield estimation, 14.73% and 17.26% higher than reference images from early or late in the season, respectively. (3) The accuracy of the yield estimation model of the three plots is different and relates to the MDI of the plots and the types of surrounding crops. These results can be used as a reference for data fusion for vegetation monitoring using remote sensing at the field scale.