CDK4/6 inhibitors (CDK4/6i) improve outcome in patients with advanced estrogen receptor-positive, HER2− breast cancer. The phase 3 SONIA trial compared the addition of CDK4/6i to first- versus second-line endocrine therapy for time to disease progression after second-line treatment (progression-free survival after two lines of treatment (PFS2)), as well as for secondary outcomes overall survival, PFS after one line of treatment (PFS1), health-related quality of life (HRQOL), toxicity and cost-effectiveness. No significant difference in PFS2 was observed; however, on an individual patient level this may be different. Using prespecified circulating tumor DNA analyses, we performed an exploratory study to evaluate whether pretreatment circulating tumor DNA (ctDNA) levels in plasma can identify patients that benefit from CDK4/6i during their first-line treatment. Cell free DNA before start of first-line treatment from 409 female patients participating in SONIA was analyzed with the modified fast aneuploidy screening test-sequencing system. This assay yields a genome-wide aneuploidy score, indicative of ctDNA levels. Cox proportional hazard analyses for PFS1 and PFS2 were performed separately for the ctDNA high group (aneuploidy score ≥ 5) and the ctDNA low group (aneuploidy score < 5). In total, 141 of the 409 included patients had a high genome-wide aneuploidy score at baseline. PFS2 in the first- compared to the second-line CDK4/6i strategy showed hazard ratios of 0.58 (95% confidence interval 0.38–0.88) and 1.36 (95% confidence interval 0.95–1.96) in the high and low aneuploidy group, respectively. A significant interaction was demonstrated between treatment strategy and aneuploidy score for PFS2 (P = 0.004). In conclusion, this study demonstrated that pretreatment ctDNA levels can be used to identify patients that benefit from first-line CDK4/6i treatment. ClinicalTrials.gov registration: NCT03425838.