Heterosis has been widely used to increase grain quality and yield, but its genetic mechanism remains unclear. In this study, the genetic basis of heterosis for four maize kernel traits was examined in two test populations constructed using a set of 184 chromosome segment substitution lines (CSSLs) and two inbred lines (Zheng58 and Xun9058) in two environments. 63 and 57 different heterotic loci (HL) were identified for four kernel traits in the CSSLs × Zheng58 and CSSLs × Xun9058 populations, respectively. Of these, nine HL and six HL were identified for four kernel traits in the CSSLs × Zheng58 and CSSLs × Xun9058 populations, at the two locations simultaneously. Comparative analysis of the HL for the four kernel traits identified only 21 HL in the two test populations simultaneously. These results showed that most HL for the four kernel traits differed between the two test populations. The common HL were important loci from the Reid × Tangsipingtou heterotic model, and could be used to predict hybrid performance in maize breeding.