Recent studies suggest that most cases of myelodysplastic syndrome (MDS) are clonally heterogeneous, with a founding clone and multiple subclones. It is not known whether specific gene mutations typically occur in founding clones or subclones. We screened a panel of 94 candidate genes in a cohort of 157 patients with MDS or secondary acute myeloid leukemia (sAML). This included 150 cases with samples obtained at MDS diagnosis and 15 cases with samples obtained at sAML transformation (8 were also analyzed at the MDS stage). We performed whole-genome sequencing (WGS) to define the clonal architecture in eight sAML genomes and identified the range of variant allele frequencies (VAFs) for founding clone mutations. At least one mutation or cytogenetic abnormality was detected in 83% of the 150 MDS patients and 17 genes were significantly mutated (false discovery rate⩽ 0.05). Individual genes and patient samples displayed a wide range of VAFs for recurrently mutated genes, indicating that no single gene is exclusively mutated in the founding clone. The VAFs of recurrently mutated genes did not fully recapitulate the clonal architecture defined by WGS, suggesting that comprehensive sequencing may be required to accurately assess the clonal status of recurrently mutated genes in MDS.