Deletions in the AZFb region of the Y chromosome contribute to male infertility; however, the clinical significance of different deletion architectures remains insufficiently resolved by conventional sequence-tagged site analysis. In this study, four men carrying AZFb deletions were analysed by multiplex polymerase chain reaction, Nanopore long-read sequencing, and modified multiplex ligation-dependent probe amplification. Nanopore long-read sequencing enabled high-resolution mapping of deletion boundaries and comprehensive characterisation of coding gene loss across the region. Distinct deletion architectures involving RBMY, EIF1AY, HSFY, KDM5D, PRY and RPS4Y2 were identified and showed potentially different associations with spermatogenic phenotypes. One case carried a complete deletion and exhibited non-obstructive azoospermia (NOA). Two cases with partial deletions retained subsets of coding genes and exhibited residual spermatogenesis, enabling successful intracytoplasmic sperm injection outcomes. Notably, one deletion initially classified as proximal partial deletion by sequence-tagged site analysis was shown by Nanopore long-read sequencing to remove all annotated coding genes, and was associated with NOA and failed testicular sperm retrieval. These results demonstrate that Nanopore long-read sequencing refines sequence-tagged site-defined AZFb deletions through delineation of deletion intervals and gene content, may providing an improved assessment of spermatogenesis in male infertility.