Background: Recent evidence has suggested that some patients with non-small cell lung cancer (NSCLC) harbor a HRD signature that represents a distinct genomic subtype that could be targeted by PARP inhibitors (PARPi). However, there is little data on HRD prevalence in NSCLC or its genomic associations. Here, we evaluated the co-occurrence of driver mutations and established immune biomarkers with an RNA-based HRD signature in a large, real-world NSCLC cohort. Methods: We analyzed data from 5119 NSCLC patients that underwent sequencing via the Tempus xT test (DNA-seq of 648 genes; RNA-seq with whole exome capture). HRD status was predicted by the Tempus HRD-RNA test, a pan-cancer logistic regression classifier that uses an RNA gene expression signature optimized to distinguish between BRCA-biallelic loss and homologous recombination repair (HRR)-WT samples (Leibowitz et al, 2022). Cohort samples were excluded from model training. All comparisons were tested via chi-squared or Fisher’s exact tests. Results: An RNA-derived signature of HRD (HRD-RNA+) was observed in 3.53% (n=181/5119) of patients. HRD-RNA+ prevalence was higher in squamous cell carcinoma (84/1331, 6.3%) relative to adenocarcinoma (68/3015, 2.3%; p < 0.001). The prevalence of select alterations by HRD-RNA status are shown in Table. Alterations in BRCA1/2 and HRR genes (inclusive of BRCA1/2) were enriched in HRD-RNA+ vs. HRD-RNA- cases (8.8% vs. 2.5%, p < 0.001; 22% vs. 15%, p = 0.008 respectively). Notably, 141 (78%) HRD-RNA+ patients had no alterations in HRR genes. Of all NCCN targetable driver mutations assessed, KRAS G12C and ALK fusions were the only targetable drivers with significantly different prevalence in HRD-RNA+ vs. HRD-RNA- patients. Across the entire cohort, NCCN driver mutations were depleted in HRD RNA+ patients (18% in HRD-RNA+ vs. 30% in HRD-RNA-, p < 0.001). Immune biomarkers (TMB, PD-L1) did not vary by HRD-RNA status. Conclusions: Compared to HRD-RNA- NSCLC, HRD-RNA+ NSCLC represents a unique, molecularly defined subset that has a decreased prevalence of NCCN-driver mutations and is not enriched for TMB-H or PD-L1 expression. Further, this signature increases the number of patients classified as HRD-RNA+ compared to HRR gene alterations alone. Functional characterization (e.g. RAD51 foci immunofluorescence assay) and clinical benefit of targeted HRD therapies such as PARPi should be explored in this HRD-RNA+ population.