Prolonged cannabis use has a significant impact on health and well-being. Genetic factors are known to influence cannabis dependence, but few specific genetic markers have been identified. ABCB1 polymorphisms are known to modify drug pharmacokinetics but have yet to be studied for their role in generating and maintaining cannabis dependence. The objective of this study is to determine if ABCB1 C3435T polymorphism may represent an independent genetic marker for cannabis dependence risk. An open bi-centric association study was conducted in two French Addiction Centres. Caucasian patients diagnosed with isolated cannabis dependence were compared with healthy age-matched controls for socio-demographic, clinical and genetic data using chi-square test, Fisher's exact test, or Mann-Whitney U test. Independent association between ABCB1 C3435T SNP marker and cannabis dependence was evaluated using multiple logistic regression analysis. Versus controls (n=40), patients with cannabis dependence (n=40) had a significantly higher 3435C allele frequency (62.5% versus 43.8% respectively, P=0.017) and CC genotype (50% versus 20%, P=0.005, OR=4.00 [1.50-10.60]). Multiple logistic regression analysis of the C3435T SNP and variables identified in univariate analyses indicated that the CC genotype was independently associated with cannabis dependence (P=0.045, OR=6.61 [1.05-46.58]). This is the first time a significant specific genetic marker has been shown in cannabis dependence. ABCB1 polymorphisms may alter Delta9THC distribution, its psychoactive effects and individual vulnerability to dependence. These results pave the way to a new pharmacogenetic hypothesis in cannabis dependence.