Background Autism Spectrum Disorders (ASD) comprise a heterogeneous group of highly heritable (50%) neurodevelopmental disorders with a multi-factorial origin. Diagnoses of ASD is mainly based on i) deficiencies in social interaction and communication and ii) restrictive and repetitive behavioral patterns, interests or activities, including sensorial alterations. However, the diagnostic criteria has limited use in research field, as different subtypes have been identified within ASD. In addition to important polygenic component, a great variety of single-gene disorders and chromosomal abnormalities have been described across latest genetic studies. Proper biological pathways and/or specific physiopathological mechanisms involved such as chromatin remodeling, glutamate/GABA balance or transcription processes have been identified as candidate gene sets enriched in deleterious rare, inherited and de novo variation. Here we present phenotypic clusters identified from a sample of well-characterized ASD trios. We describe distinctive biological pathways involved and described types of functional variation related with mentioned clusters. Methods Our sample has 250 Spanish ASD trios recruited at Hospital Universitario Gregorio Maranon. Demographic, clinical and neuropsychological variables were collected from trio samples to perform cluster analysis with a combination of hierarchical and k-mean methodology. General linear model was used to study association between genetic and phenotypic variables collected. Exome from blood DNA was sequenced as part of the Autism Sequencing Consortium (ASC) dataset. ANNOVAR was used for variant annotation from vcf files to filter rare (MAF Results ASD sample was exhaustively characterized, and clusters were defined according to several variables as Intellectual Disability (ID), developmental regression, psychiatric comorbility, gastrointestinal pathology or pro-inflamatory status. Distinctive patterns of protein-truncating, damaging missense, de novo, inherited ultrarrare or intolerant variation across identified phenotypic clusters were found, affecting previously implicated gene sets as chromatin remodeling, synapse and immune-related pathways. Interestingly, in preliminary results we found a clear connection between immune-affected cluster (defined by phenotype and biomarkers) and intestinal membrane adhesion genes. Discussion Through the study here presented, we demonstrated enrichment of functional genetic rare variation of distinctive nature across identified phenotypic clusters in our dataset, deepening previous findings in relation with different entities within ASD. We claim the usefulness of differentiate biological clusters with diagnosable attributes as this aim will not only allow the establishment of stratification criteria for clinical purposes, but also improve decision making about pharmacological treatment choice and strengthen the importance of early intervention therapies.