Individuals with autism spectrum disorder (ASD) perform poorly in working memory (WM) tasks, with some literature suggesting that their impaired performance is modulated by WM load. While some neuroimaging and neurophysiological studies have reported altered functional connectivity during WM processing in individuals with autism, it remains largely unclear whether such alterations are moderated by WM load. The present study aimed to examine the effect of WM load on functional connectivity within the prefrontal cortex (PFC) in ASD using functional near-infrared spectroscopy (fNIRS). Twenty-two children with high-functioning ASD aged 8–12 years and 24 age-, intelligent quotient (IQ)-, sex- and handedness-matched typically developing (TD) children performed a number n-back task with three WM loads (0-back, 1-back, and 2-back). Hemodynamic changes in the bilateral lateral and medial PFC during task performance were monitored using a multichannel NIRS device. Children with ASD demonstrated slower reaction times, specifically during the “low load” condition, than TD children. In addition, the ASD and TD groups exhibited differential load-dependent functional connectivity changes in the lateral and medial PFC of the right but not the left hemisphere. These findings indicate that WM impairment in high-functioning ASD is paralleled by load-dependent alterations in right, but not left, intrahemispheric connectivity during WM processing in children with ASD. A disruption of functional neural connections that support different cognitive processes may underlie poor performance in WM tasks in ASD. Copyright © 2022 The Author(s).