Prostate cancer (PCa) frequently presents as a multifocal disease within a single gland. Herein, the transcriptome-wide profiles of glandular epithelial (GE) cells of four PCa tissues with various Gleason scores (GSs) are analyzed with Visium spatial transcriptomics (ST). The genetic classifications across PCa section sites generally matched the spatial patterns of histological structures with different GSs. Average inferred copy number variation (inferCNV) values gradually increased during GS development. Developing trajectories during GS upgrading were assessed, and differentially expressed genes (DEGs) during GS progression were analyzed which exhibited heterogeneity among individual patients with PCa. Several crucial genes, such as NANS, PABPC1L, PILRB, PPFIA2, and SESN3, were associated with GS upgrading. Enrichment analysis showed that biological functions, such as cadherin binding, Golgi vesicle transport, protein folding, and cell adhesion molecules were related to GS progression. In conclusion, this study provides insight into ST-based transcriptome-wide expression patterns during GS progression.© 2023 The Author(s).