Single gene mutations leading to obesity though rare have provided critical insights into the molecular and physiological mechanisms underlying control of energy homeostasis and body weight. No systematic studies have been carried out to assess the genetic spectrum of extremely obese cases (SDS > 3) in a predominantly consanguineous population. In the present study we searched for obesity-associated mutations by a multi-layered combination of sequencing techniques in a cohort of 175 unrelated subjects with early onset severe obesity and their family members, from a Pakistani consanguineous population. All subjects were, initially, screened for mutations in of leptin (LEP) and melanocortin 4 receptor (MC4R) genes by Sanger sequencing. Subjects negative for mutations in these genes were screened for 27 obesity-associated genes using microdroplet PCR-enrichment followed by next generation sequencing (NGS). Genomic structural variations were assessed by genome-wide genotyping. Forty severely obese children and family members in whom mutations in known obesity-associated genes could not be identified using the aforementioned procedures were analysed by whole exome sequencing (WES). Hormone levels were analysed by immunoassays. Using Sanger and microdroplet-based sequencing, and genotyping, we identified 54 probands carrying 20 different homozygous loss-of-function mutations in 7 genes: LEP (n=35), leptin receptor (LEPR) (n=11), MC4R (n=4), Bardet-Biedl syndrome (BBS) (n=4) and Prader-Willi syndrome (PWS) (n=2). We also found a ~59 kb deletion in chromosome 1, encompassing LEPR. Lastly, WES revealed potentially pathogenic mutations in three susceptible genes, insulin induced gene 2 (INSIG2), Rho-associated protein kinase 1 (ROCK1), adenylyl cyclase 3 (ADCY3). Link of these genes to obesity is further supported by previous GWASs and/or animal studies. Notably, leptin values were significantly increased in LEPR deficient and cortisol levels raised in leptin deficient subjects as compared to those with MC4R mutations. Furthermore, gut hormones were shown to play a minimal role in inducing hyperphagia in subjects with congenital deficiency. A high prevalence (32%) of pathogenic mutation in this population, underscores the importance of comprehensive genetic screening of inbred populations to unravel new genes and signalling pathways modulating energy balance and providing leads to evidence-based patient management and wherever possible personalized treatment.