Objective: Poor physiological regulation in response to threat is linked to multiple negative developmental outcomes including anxiety, which is highly prevalent and impairing in young children with neurodevelopmental disabilities like fragile X syndrome (FXS) and autism spectrum disorder (ASD). The present study contrasted cardiac startle response in pre-school-aged children with FXS, with and without ASD, to children with non-syndromic ASD (nsASD) and neurotypical controls (NT). The relationship of cardiac startle to non-verbal mental age (NVMA), ASD severity, and parent-reported anxiety was also examined. Method: Four age-matched groups of pre-school children participated including those with FXS without ASD (FXS-Only, n = 21), FXS with ASD (FXS+ASD, n = 17), nsASD (n = 42), and NT children (n = 27). Participants viewed a silent movie during which a single 200 ms 98-decibel white noise burst occurred. Cardiac activity was analyzed for pre-stimulus respiratory sinus arrhythmia (RSA) and the inter-beat intervals (IBI) at the auditory stimulus and 10 s post-stimulus. The Spence Pre-school Anxiety Scale, Autism Diagnostic Observation Schedule-2nd Edition, and Mullen Scales of Early Learning were examined in relation to startle response. Results: The nsASD group demonstrated heightened cardiac activity at the auditory stimulus and 10 s post-stimulus compared to the NT controls. Neither of the FXS groups showed differences from any other group. Higher pre-stimulus RSA was associated with reduced cardiac response across groups, while the relationship between cognitive ability and ASD severity to cardiac response varied between groups. Parent-reported anxiety was not associated with cardiac response for any group. Conclusion: These findings demonstrate group distinctions in cardiac responses to auditory startle. Although FXS and ASD share behavioral characteristics, the nsASD group showed a heightened cardiac startle response compared to the NT group that was not present in the FXS groups with or without ASD. Non-verbal mental age was associated with greater stimulus or post-stimulus reactivity for all groups except the FXS+ASD group, which showed no association between startle response and any clinical outcomes. Increased understanding of the relationship between physiological regulation and clinical outcomes will assist in identifying the timing and targets for effective interventions for individuals with neurodevelopmental disabilities.