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Abstract Summary Tumor heterogeneity has emerged as a fundamental property of most human cancers, with broad implications for diagnosis and treatment. Recently, spatial omics have enabled spatial tumor profiling, however computational resources that exploit the measurements to quantify tumor heterogeneity in a spatially aware manner are largely missing. We present ATHENA (Analysis of Tumor HEterogeNeity from spAtial omics measurements), a computational framework that facilitates the visualization, processing and analysis of tumor heterogeneity from spatial omics measurements. ATHENA uses graph representations of tumors and bundles together a large collection of established and novel heterogeneity scores that quantify different aspects of the complexity of tumor ecosystems. Availability and implementation ATHENA is available as a Python package under an open-source license at: https://github.com/AI4SCR/ATHENA. Detailed documentation and step-by-step tutorials with example datasets are also available at: https://ai4scr.github.io/ATHENA/. The data presented in this article are publicly available on Figshare at https://figshare.com/articles/dataset/zurich_pkl/19617642/2. Supplementary information Supplementary data are available at Bioinformatics online.