Melanocortin-4 receptor (MC4R) loss-of-function mutations are the commonest genetic cause of human monogenic obesity, so far. The contribution of MC4R coding mutations to severe obesity in the high-obesity prone Greek population has not been investigated to date. We determined the MC4R coding sequence of 510 obese and 469 lean control subjects of Greek origin, and we estimated the prevalence and the penetrance on obesity of MC4R loss-of-function mutations. The functional impact of novel nonsynonymous variants detected was investigated in vitro. We found two novel synonymous mutations (L23L and I102I), four nonsynonymous mutations (T112M, S127L, N274S, and S295L), and two polymorphisms (V103I and I251L) previously described in literature. We also detected a novel mutation (L207V) in a severely obese 69-year-old female patient, although the mutation did not cosegregate with obesity in the corresponding pedigree and had no functional consequences on MC4R protein function. Loss-of-function mutations represented 75% of all nonsynonymous rare mutations identified among lean carriers and only 25% among obese subjects (P = 0.0001). The prevalence of loss-of-function mutations was lower in the obese group than in lean control subjects (0.20 vs. 0.64%) but this difference was not significant. Therefore, the estimated penetrance of deleterious MC4R mutations was very low (6.3%) in heterozygous Greek carriers of MC4R loss-of-function mutations. Our data suggest that MC4R loss-of-function mutations are rare in the Greek population. MC4R genetic deficiency is unlikely to explain the high propensity to develop severe obesity in this specific population.