Vascular malformations are part of overgrowth syndromes characterized by somatic mosaic mutations or rarely by germline mutations. Due to their similarities and diversity, clinicopathological classification can be challenging. A comprehensive targeted Next Generation Sequencing screen using Unique Molecular Identifiers (UMI's) with a technical sensitivity of 1% mutant alleles was performed for frequently mutated positions in ≥21 genes on 319 formalin-fixed paraffin-embedded samples. In 132 out of 319 cases (likely) pathogenic mosaic mutations were detected affecting genes previously linked to vascular malformations e.g. PIK3CA (n=80), TEK (TIE2) (n=11), AKT1 (n=1), GNAQ (n=7), GNA11 (n=4), IDH1 (n=3), KRAS (n=9), and NRAS (n=1). Six cases harbored a combination of pathogenic mutations in PIK3CA and in GNA11 (n=2), GNAQ (n=2), or IDH1 (n=2). Aberrations in PTEN and RASA1 with a variant allele frequency approaching 50% suggestive of germline origin were identified in six out of 102 cases tested; four contained a potential second hit at a lower allele frequency. Ninety-one of the total 142 (likely) pathogenic mutations were present at a variant allele frequency <10% illustrating the importance of sensitive molecular analysis. Clinicopathological characteristics showed a broad spectrum and overlap when correlated with molecular data. Sensitive screening of recurrently mutated genes in vascular malformations may help to confirm the diagnosis and reveals potential therapeutic options with a significant contribution of PIK3CA/mTOR and RAS-MAPK pathway mutations. The co-existence of two activating pathogenic mutations in parallel pathways illustrates potential treatment challenges and underlines the importance of multigene testing. Detected germline mutations have major clinical impact. This article is protected by copyright. All rights reserved.