Multispectral (MS) imaging enables the measurement of characteristics important for increasing the prediction accuracy of genotypic and phenotypic values for yieldrelated traits. In this study, we evaluated the potential application of temporal MS imaging for the prediction of aboveground biomass (AGB) in soybean [Glycine max (L.) Merr.]. Field experiments with 198 accessions of soybean were conducted with four different irrigation levels. Five vegetation indices (VIs) were calculated using MS images from soybean canopies from early vegetative to early reproductive stage. To predict the genotypic values of AGB, VIs at the different growth stages were used as secondary traits in a multitrait genomic prediction. The prediction accuracy of the genotypic values of AGB from MS and genomic data largely outperformed that of the genomic data alone before the flowering stage (90% of accessions did not flower), suggesting that it would be possible to determine cross-combinations based on the predicted genotypic values of AGB. We compared the prediction accuracy of a model using the five VIs and a model using only one VI to predict the phenotypic values of AGB and found that the difference in prediction accuracy decreased over time at all irrigation levels except for the most severe drought. The difference in the most severe drought was not as small as that in the other treatments. Only the prediction accuracy of a model using the five VIs in the most severe droughts gradually increased over time. Therefore, the optimal timing for MS imaging may depend on the irrigation levels.Abbreviations: AGB, aboveground biomass; DTF, days to flowering; GEI, genotype × environment interaction; GRVI, green-red vegetation index; MS, multispectral; MT, multitrait; NDI red , normalized difference index using red-edge and red spectral reflectance; NDRE, normalized difference red-edge index; NDVI, normalized difference vegetation index; PCA, principal component analysis; RTVI, red-edge triangular vegetation index; SNP, single-nucleotide polymorphism; UAV, unmanned aerial vehicle; VI, vegetation index.This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.