Background: The exposure to endocrine therapy (ET) can induce the onset of ESR1 gene alterations that have an impact on not only treatment resistance but also clinical phenotype. We previously demonstrated the potential of liquid biopsy in describing the metastatic behavior of MBC. The aim of this study was to explore the different clinical phenotype across the main ESR1 and PIK3CA codon variants. Methods: The study retrospectively analyzed a cohort of 501 MBC patients (pts) characterized for ctDNA through NGS before treatment start at Northwestern University (Chicago, IL), Massachusetts General Hospital (Boston, MA), CRO National Cancer Institute (Aviano, IT) and ASUFC Hospital (Udine, IT) between 2014 and 2020. Associations between clinical characteristics and ESR1 and PIK3CA codon variants were explored through logistic regression corrected for sites and ESR1/PIK3CA status. Survival was tested through Cox regression both for progression-free survival (PFS) and overall survival (OS). Results: Of the total 501 pts, 289 (58%) were diagnosed with hormone-receptor positive (HRpos) MBC, 114 (23%) with HER2-positive MBC, and 93 (19%) with triple-negative MBC. ESR1 mutations were detected in 71 pts (14%) and PIK3CA in 154 pts (31%). The most represented ESR1 gene mutations were found in codons 380 (9%), 536 (23%), 537 (34%), and 538 (34%), while alterations in codons 542 (19%), 545 (21%), and 1047 (60%) were the most common for PIK3CA. As expected, ESR1 mutations were found only in HRpos pts previously exposed to ET (P < 0.001). No significant differences were observed for PIK3CA. After multivariable analysis, ESR1mutations were confirmed as highly associated with liver and bone metastases (OR 3.31, P < 0.001 and OR 5.09, P < 0.001). Moreover, an association with lung (OR 2.07, P = 0.010) was observed in this cohort. After multivariable analysis, codon 537 mutations were associated with bone involvement (OR 12.97, P = 0.014), codon 538 with liver (OR 4.73, P = 0.010), and codon 536 with soft tissue (OR 5.84, P = 0.006) and liver (OR 4.06, P = 0.048). PIK3CA mutations were associated with bone (OR 2.61, P < 0.001) and lung metastases (OR 1.62, P = 0.044). Specifically, codon 1047 mutations were the primary driver (OR 3.14, P = 0.001 and OR 1.97, P = 0.019). In HRpos MBC, baseline mutations in ESR1 codon 537 and 538 had a negative impact on OS (HR 3.73, P < 0.010 and HR 2.99, P < 0.021), while 380 and 536 had a negative impact on PFS (HR 18.98, P < 0.001 and HR 2.60, P = 0.015). No impact was observed across PIK3CA gene variants. Conclusions: This study showed the different tumor biology across ESR1 and PIK3CA gene variants. As novel selective estrogen receptor degraders (SERDS) and PIK3CA inhibitors are gaining momentum as new ET options in MBC, these results highlight the future pivotal role of ctDNA NGS in refining tumor biology characterization.