The diagnosis of Autism Spectrum Disorder (ASD) relies on history and behavioral observation, lacking reliable biomarkers. We performed a retrospective analysis using machine learning algorithms of 928 persons with ASD (mean age: 17 ± 10.8 years; age range 4-67) obtained from a multisite psychiatric database with rest and on-task brain SPECT scans to investigate whether or not these scans distinguish ASD from healthy controls (HC, n=101; mean age: 43 ± 17.2 years; age range 13-84). Using 128 regions of interest extracts (ROIs), we applied multiple machine learning algorithms for binary classification. Due to an unbalanced sample size between ASD and controls, we then sub-sampled the data prior to feature selection and classification. Using a subsampled dataset, least absolute shrinkage and selection operator (LASSO) feature selection with Random Forest method baseline accuracy results of approximately 81% were achieved, based on optimal classifier settings with the top selected features. We applied machine learning algorithms to ASD adults only, the majority of our sample, and selected subjects in both AD and HC groups with age range of 13-67 years and found the results consistent with the combined data. These machine learning results identified potential diagnostic biomarkers differentiating ASD from HC in the regions of the cerebellum and vermis, anterior cingulate gyrus, amygdala, thalamus, frontal, and temporal lobes.