Background: ROS1 is an important proto-oncogene involved in the development of various cancers for which we have FDA approved therapies. While activation of the ROS1 tyrosine kinase receptor has been reported in 1-2% of lung cancers, the frequency and type of ROS1 alterations in breast cancer have not been fully explored. We previously described the incidence of ROS1 alterations from breast cancer tissue. The purpose of this study was to identify the incidence of ROS1 genomic alterations occurring in cfDNA from patients with breast cancer. Methods: We queried 16,053 breast cancer samples from the Guardant Health breast cancer database between June 2015 - October 2019 to identify the incidence of ROS1 alterations detected in cfDNA in breast cancer. We identified fusion partner genes and classified each alteration type into the following categories: fusion, single nucleotide variants (SNVs), and indels. Radical amino acid changes occurring at conserved regions across the ROS1 gene were identified. In vitro analyses were used to investigate the effect of ROS1 nonsynonymous mutations on the ROS1 protein. We made associations with ROS1 alterations and co-occurring mutated genes. Results: Nonsynonymous ROS1 alterations from the Guardant Health breast cancer database were found in 162 samples from 142 patients in the 16,053-patient cohort (1%). Alterations found included: 1 (0.6%) ROS1-SLC35F1 fusion, 155 (95.7%) SNVs, and 6 (3.7%) indels. Of the 155 SNVs, we identified 23 (14.8%) mutations occurring in the ROS1 kinase, of which, 20 (12.9%) occurred at highly conserved regions and 15 (9.6%) harbored radical amino acid changes. The top 5 co-occurring mutations in samples with ROS1 alterations were TP53 (50%), PIK3CA (44%), ESR1 (27%), EGFR (21%), and FGFR1 (18%). Conclusions: A modest incidence of ROS1 genomic alterations occurs in cfDNA from patients with breast cancer. New somatic alterations in the ROS1 gene were identified from Guardant Health that were not detected in publicly available databases. A portion of mutations occurred at highly conserved regions across the ROS1 gene suggesting these may be more actionable than currently recognized. In vitro analyses of ROS1 gene activation from these newly discovered somatic alterations are being investigated with results to be reported. Co-occurring mutations reveal a unique genotype associated with ROS1 alterations that may play a biologic role in ROS1-mediated pathogenesis.