Whole-transcriptome spatial profiling of genes at single-cell resolution remains a challenge. To address this limitation, spatial gene expression prediction methods have been developed to infer the spatial expression of unmeasured transcripts, but the quality of these predictions can vary greatly. Here we present TISSUE (Transcript Imputation with Spatial Single-cell Uncertainty Estimation) as a general framework for estimating uncertainty for spatial gene expression predictions and providing uncertainty-aware methods for downstream inference. Across eleven benchmark datasets, TISSUE provides well-calibrated prediction intervals for predicted expression values. Moreover it consistently reduces false discovery rates for differential gene expression analysis, improves clustering and visualization of predicted spatial transcriptomics, and improves the performance of supervised learning models trained on predicted gene expression profiles. Applying TISSUE to a MERFISH spatial transcriptomics dataset of the adult mouse subventricular zone, we identified subtypes within the neural stem cell lineage and developed subtype-specific regional classifiers. TISSUE is publicly available as a flexible wrapper method for existing spatial gene expression prediction methods to assist researchers with implementing uncertainty-aware analyses of spatial transcriptomics data.