Heterosis can increase the yield of many crops and has been extensively applied in agriculture. In maize, female inflorescence architecture directly determines grain yield. Thus, exploring the relationship between early maize ear inflorescence development and heterosis regarding yield-related traits may be helpful for characterizing the molecular mechanisms underlying heterotic performance. In this study, we fine mapped the overdominant heterotic locus (hlEW2b), associated with ear width, in an approximately 1.98-Mb region based on analyses of chromosome segment substitution lines and the corresponding testcross population. Maize ear inflorescences at the floral meristem stage were collected from two inbred lines, one chromosome segment substitution line that carried hlEW2b (sub-CSSL16), the receptor parent lx9801, and the Zheng58 × sub-CSSL16 and Zheng58 × lx9801 hybrid lines. A total of 256 metabolites were identified, including 31 and 24 metabolites that were differentially accumulated between the two hybrid lines and between the two inbred lines, respectively. Most of these metabolites are involved in complex regulatory mechanisms important for maize ear development. For example, nucleotides are basic metabolites affecting cell composition and carbohydrate synthesis. Additionally, nicotinate and nicotinamide metabolism is important for photosynthesis, plant stress responses, and cell expansion. Moreover, flavonoid and phenolic metabolites regulate auxin transport and cell apoptosis. Meanwhile, phytohormone biosynthesis and distribution influence the cell cycle and cell proliferation. Our results revealed that changes in metabolite contents may affect the heterotic performance related to ear width and yield in maize hybrid lines. This study provides new clues in heterosis at the metabolomics level and implies that differentially accumulated metabolites made distinct contributions to the heterosis at an early stage of ear inflorescences development.