Abstract Atopic dermatitis (AD) is a chronic, multifactorial inflammatory disorder characterized by skin barrier defects and immune dysregulation. Despite substantial evidence implicating genetic factors in its pathogenesis, including the role of genes like FLG and HLA, the exact genetic mechanisms underlying AD remain elusive. Whole-exome sequencing (WES) offers a promising avenue for identifying rare and common genetic variants associated with AD. This study aims to explore the utility of WES in elucidating the genetic basis of AD within an Indian cohort, where data are currently limited. The study was conducted at a paediatric dermatology clinic, recruiting 16 patients with AD based on the Hanifin-Rajka criteria. Clinicodemographic details, Scoring Atopic Dermatitis scores and serum IgE levels were recorded. WES was performed on DNA isolated from blood samples using the Twist Biosciences Human Comprehensive Exome Panel, followed by Illumina sequencing. Bioinformatic ana­lysis included variant alignment, annotation and filtering through ANNOVAR. Variants were further analysed for AD-related genes and compared with an in-house database of 2438 controls. Statistical significance was assessed using the χ2-test, with a threshold of P < 0.05. The cohort had a mean age of 9.59 years (SD 3.15); six patients were female and 10 male). They had a mean disease onset age of 36.4 months (SD 33.3) and a mean IgE level of 6564 IU mL−1. WES identified significant mutations in key genes, including FLG and HLA, with variant frequencies significantly higher in cases than controls (P < 0.001). Notable findings include the following. (i) In FLG (chr1), multiple loss-of-function variants, such as 152276761T>C and 152276828G>A, were present in 63% of cases. (ii) In HLA-A, HLA-DQA1 and HLA-DQB1 (chr6), variants such as 29911928C>G (81%) and 32609299A>- (44%) were prominent. These findings corroborate the role of epidermal differentiation complex genes and immune regulatory genes in AD pathogenesis. This study highlights the utility of WES in uncovering genetic variants implicated in AD, particularly in an Indian cohort. Consistently with global studies, FLG loss-of-function mutations were associated with higher IgE levels and earlier disease onset. Novel findings, including the prevalence of HLA variants, expand the understanding of genetic predisposition in AD. The study underscores the potential of WES in identifying ethnicity-specific genetic factors and facilitating personalized therapeutic strategies. In conclusion, WES provides invaluable insights into the genetic architecture of AD. This pilot study emphasizes the need for larger, multicentric studies to further elucidate the genetic landscape of AD in diverse populations, paving the way for personalized medicine.