Background: Child growth results from the interaction of genetic, endocrine, nutritional, and environmental factors. Short stature is defined as a height-for-age Z-score below −2 standard deviations, according to the WHO Child Growth Standards 2006. Linear bone growth occurs through endochondral ossification, which is influenced by insulin-like growth factor-1 (IGF-1), 25-hydroxyvitamin D [vitamin 25(OH) D], calcium (Ca), phosphorus (Ph), and magnesium (Mg). This study compared these biomarkers across growth phenotypes and examined their associations with short stature in children aged 24–59 months. Methods: This cross-sectional analytical study used registry data and stored biological specimens (SBS) from 225 children aged 24–59 months residing in Bandung District, Indonesia (84 underweight children with short stature, 70 normal-weight children with short stature, and 71 children with normal weight and stature), collected between May and August 2021. SBS were preserved at -80 °C. Appropriate statistical tests, post-hoc analyses, and multivariable logistic regression were performed. Results: Significant differences in IGF-1, 25(OH)D, calcium, phosphorus, and magnesium were observed across groups (all p < 0.05). Post-hoc analysis indicated that these differences were confined to comparisons between the underweight short stature group and the normal-weight normal stature group, with no significant differences involving the normal-weight short stature group. Multivariable analysis further showed that lower levels of IGF-1, calcium, phosphorus, and magnesium were independently associated with short stature, with these associations largely attributable to the underweight subgroup, while remaining non-significant among children with normal-weight short stature. Conclusion: Lower levels of IGF-1, calcium, phosphorus, and magnesium are associated with short stature in children aged 24–59 months, predominantly among those with concurrent undernutrition. In contrast, these associations were not evident in children with normal-weight short stature, underscoring the heterogeneous nature of short stature.