Changes in the desaturation activity of LC-PUFAs may influence insulin sensitivity by modulating the relative abundance of omega-3. The aim of this cross-sectional study was to assess the association between genetic variants of fatty acid desaturase cluster genes (FADS1, FADS2, FADS3) and insulin sensitivity in a cohort of children and adolescents with obesity. Anthropometric evaluation, lipid profile, glucose metabolism parameters and the genotype of rs1535 on FADS2 gene were assessed. In 162 obese children and adolescents (12.6 ± 2.3 years; BMI 30.9 ± 7.3), we found a significant association between an index of insulin sensitivity, i.e., Matsuda index, and rs1535 (B = −0,192; p = 0.008), BMI (B = −0,003; p < 0.001), and triglycerides (B = −0,034; p < 0.001), independent of age and sex [R² = 0.35; p = <0.001]. In conclusion, FADS cluster variants were associated with insulin sensitivity in a population of children and adolescents with obesity, contributing to identify individuals who may benefit from personalised prevention and treatment nutritional strategies since childhood.