<span class=”paragraphSection”><div class=”boxTitle”>Abstract</div><div class=”boxTitle”>Background:</div>Numerous studies have sought to identify genes that influence the ability to quit smoking, but none found any that are consistently associated with smoking cessation.<div class=”boxTitle”>Methods:</div>We developed a novel difficulty of quitting smoking phenotype based on the extremes of the number of quit attempts needed to achieve successful abstinence: Easy quitters were defined as having achieved long-term (>1 year) abstinence after their first quit attempt and difficult quitters as having reported 10 or more quit attempts. We conducted a two-stage study to determine if this phenotype could be useful for identifying single nucleotide polymorphisms (SNPs) that influence smoking cessation. In stage 1, 82 SNPs in 26 genes involved in nicotine signaling and metabolism were genotyped in 1357 easy quitters and 1321 difficult quitters from Cancer Prevention Study 3 (CPS-3). In stage 2, the 11 SNPs associated with difficult quitting in stage 1 (<span style=”font-style:italic;”>p</span> < .1) were genotyped in an independent sample of 1300 easy quitters and 1299 difficult quitters from CPS-3.<div class=”boxTitle”>Results:</div>Three of 11 SNPs (<span style=”font-style:italic;”>HTR1B</span> rs6298, <span style=”font-style:italic;”>NR4A2</span> rs834829, and <span style=”font-style:italic;”>CYP2A65</span> rs8192729) were significantly associated with the difficult quitting phenotype in stage 2 (<span style=”font-style:italic;”>p</span> < .05). In addition, a polygenic risk score based on the 11 SNPs identified in stage 1 was significantly associated with the difficult quitting phenotype in stage 2 (odds ratio = 1.08, 95% confidence interval: 1.03–1.14 per quintile, <span style=”font-style:italic;”>p</span> trend = 4.5×10<sup>–3</sup>).<div class=”boxTitle”>Conclusions:</div>Using a novel difficulty of quitting phenotype, three gene variants and a polygenic risk score based on 11 SNPs were found to be significantly associated with smoking cessation.<div class=”boxTitle”>Implications:</div>Our results provide evidence that a difficulty of quitting smoking phenotype based on the extremes of number of quit attempts could be a useful tool for identifying genetic variants that influence difficulty of smoking cessation. Knowledge of these genetic variants will indicate biological pathways that could be targeted for the development of novel smoking cessation aids and could be used to determine which smokers are most likely to benefit from such smoking cessation aids.</span>