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Plant resistance against biotic stressors is significantly influenced by pathogenesis-related 1 (PR1) proteins. This study examines the systematic identification and characterization of PR1 family genes in sugarcane (<i>Saccharum spontaneum</i> Np-X) and the transcript expression of selected genes in two sugarcane cultivars (ROC22 and Zhongtang3) in response to <i>Ustilago scitaminea</i> pathogen infection. A total of 18 <i>SsnpPR1</i> genes were identified at the whole-genome level and further categorized into four groups. Notably, tandem and segmental duplication occurrences were detected in one and five <i>SsnpPR1</i> gene pairs, respectively. The <i>SsnpPR1</i> genes exhibited diverse physio-chemical attributes and variations in introns/exons and conserved motifs. Notably, four SsnpPR1 (SsnpPR1.02/05/09/19) proteins displayed a strong protein–protein interaction network. The transcript expression of three <i>SsnpPR1</i> (<i>SsnpPR1.04/06/09</i>) genes was upregulated by 1.2–2.6 folds in the resistant cultivar (Zhongtang3) but downregulated in the susceptible cultivar (ROC22) across different time points as compared to the control in response to pathogen infection. Additionally, <i>SsnpPR1.11</i> was specifically upregulated by 1.2–3.5 folds at 24–72 h post inoculation (hpi) in ROC22, suggesting that this gene may play an important negative regulatory role in defense responses to pathogen infection. The genetic improvement of sugarcane can be facilitated by our results, which also establish the basis for additional functional characterization of <i>SsnpPR1</i> genes in response to pathogenic stress.