Autism spectrum disorder (ASD) is a common condition with lifelong implications. The last decade has seen dramatic improvements in DNA sequencing and related bioinformatics and databases. We analyzed the raw DNA sequencing files on the Variantyx® bioinformatics platform for the last 50 ASD patients evaluated with trio whole-genome sequencing (trio-WGS). “Qualified” variants were defined as coding, rare, and evolutionarily conserved. Primary Diagnostic Variants (PDV), additionally, were present in genes directly linked to ASD and matched clinical correlation. A PDV was identified in 34/50 (68%) of cases, including 25 (50%) cases with heterozygous de novo and 10 (20%) with inherited variants. De novo variants in genes directly associated with ASD were far more likely to be Qualifying than non-Qualifying versus a control group of genes (p = 0.0002), validating that most are indeed disease related. Sequence reanalysis increased diagnostic yield from 28% to 68%, mostly through inclusion of de novo PDVs in genes not yet reported as ASD associated. Thirty-three subjects (66%) had treatment recommendation(s) based on DNA analyses. Our results demonstrate a high yield of trio-WGS for revealing molecular diagnoses in ASD, which is greatly enhanced by reanalyzing DNA sequencing files. In contrast to previous reports, de novo variants dominate the findings, mostly representing novel conditions. This has implications to the cause and rising prevalence of autism.