Autism spectrum disorder (ASD) is manifested by abnormal cell numbers and patches of gene expression disruption in higher-order brain regions. Here, we investigated whether layer-specific changes in glia/neuron ratios (GNR) characterize patches in the dorsolateral prefrontal cortex (DL-PFC) of children with ASD. We analyzed high-resolution digital images of postmortem human brains from 11 ASD and 11 non-ASD children obtained from the Autism Study of the Allen Human Brain Atlas. We found the GNR is overall reduced in the ASD DL-PFC. Moreover, layers II-III belonging to patches presented a lower GNR in comparison with layers V-VI. We here provide a new insight into how brain cells are arranged within patches that contributes to elucidate how neurodevelopmental programs are altered in ASD.