The primary objective of the present study was to investigate the levels of essential trace elements in hair and serum in children with autism spectrum disorder (ASD) and investigate the age and gender effects. Children with ASD were characterized by significantly higher levels of copper (Cu) (+8%), iron (Fe) (+5%), and selenium (Se) (+13%) levels in hair and only 8% higher serum Cu levels. After stratification for gender, ASD boys were characterized by significantly increased hair Cu (+ 25%), Fe (+ 25%), and Se (+ 9%) levels, whereas in girls only Se content was elevated (+ 15%). Boys and girls suffering from ASD were characterized by significantly higher serum manganese (Mn) (+20%) and Cu (+18%) as compared to the control values, respectively. In the group of younger children (2–5 years), no significant group difference in hair trace element levels was detected, whereas serum Cu levels were significantly higher (+7%). In turn, the serum concentration of Se in ASD children was 11% lower than that in neurotypical children. In the group of older children with ASD (6–10 years), hair Fe and Se levels were 21% and 16% higher, whereas in serum only Cu levels were increased (+12%) as compared to the controls. Correlation analysis also revealed a different relationship between serum and hair trace element levels with respect to gender and age. Therefore, it is highly recommended to assess several bioindicative matrices for critical evaluation of trace element status in patients with ASD in order to develop adequate personalized nutritional correction. © 2017, Springer Science+Business Media, LLC.