Background: Cyclin-dependent kinases 4 and 6 inhibitors (CDK4 & 6i) have advanced the therapeutic landscape for patients with hormone receptor-positive (HR+), human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer (MBC). However, there is an unmet need for treatment strategies following progression on CDK4 & 6i +/- endocrine therapy (ET). The objective of this analysis is to aid in the development of post-progression treatment by characterizing genomic profiles of tumor biopsies after progression on a CDK4 & 6i +/- ET. Methods: This is a retrospective study comparing two cohorts of US patients (pts) diagnosed with HR+, HER2- MBC since January 2011. One cohort underwent biopsy following progression on a CDK4 & 6i +/- ET (Cohortpost) while the second cohort underwent biopsy with no evidence of CDK4 & 6i treatment (Cohortpre). Tumor tissue was analyzed using the next generation sequencing Tempus xT assay that analyses 595 cancer-related genes. Of 595 molecularly profiled pts, 369 had sufficient medical record data to be eligible for this analysis. Patient characteristics at metastatic diagnosis and CDK4 & 6i initiation were described. For genomic profile comparison, Mann-Whitney U-test was used for continuous variables such as tumor mutation burden (TMB) with significance at P < 0.05; Chi-square or Fishers' exact test was used as appropriate for categorical variables including mutation frequency, with significance at false discovery rate (FDR) < 0.2. Results: Of 369 pts, 177 (48%) were in Cohortpre and 192 (52%) were in Cohortpost. Overall, the mean age at the time of MBC diagnosis was 59 and 55 years, respectively; 47% and 66% of patients had evidence of prior chemotherapy or ET; and 30% and 25% had de novo stage IV MBC. The most common biopsy site was liver, occurring in 38% of pts overall (Cohortpre, 23%; Cohortpost, 52.6%). The xT assay results indicated that pts in Cohortpost had significantly higher TMB compared to patients in Cohortpre (median 2.92 vs 1.67, P < 0.0001). A subset of patients with liver mets across both cohorts (n = 141) showed a similar, but nonsignificant trend (median 2.92 vs 2.08, P = 0.0565). Additionally, pts in Cohortpost had a higher ESR1 alteration frequency compared to Cohortpre (mutations: 32.29% vs 9.60%, FDR < 0.0001; fusions: 7% vs 3%, P = 0.1420). No significant differences were noted in TP53, CCNE1, CCND1, RB1, CDK4, and CDK6 between cohorts. Conclusions: These findings describe clinical characteristics and specific genomic alterations noted after progression on CDK4 & 6i +/- ET. Further analyses will evaluate timing of resistance, mutational and transcriptomic signatures. This may aid in understanding mechanisms of progression associated with CDK4 & 6i +/- ET, ultimately contributing to development of treatment options post progression.