Dl_CO is a widely used pulmonary function test in clinical practice and a particularly useful measure for assessing patients with chronic obstructive pulmonary disease (COPD). We hypothesized that elucidating genetic determinants of Dl_CO could lead to better understanding of the genetic architecture of COPD. We estimated the heritability of Dl_CO using common genetic variants and performed genome-wide association analyses in four cohorts enriched for subjects with COPD (COPDGene [Genetic Epidemiology of COPD], NETT [National Emphysema Treatment Trial], GenKOLS [Genetics of Chronic Obstructive Lung Disease study], and TESRA [Treatment of Emphysema With a Gamma-Selective Retinoid Agonist study]) using a combined European ancestry white dataset and a COPDGene African American dataset. We assessed our genome-wide significant and suggestive associations for Dl_CO in previously reported genome-wide association studies of COPD and related traits. We also characterized associations of known COPD-associated variants and Dl_CO. We estimated the SNP-based heritability of Dl_CO in the European ancestry white population to be 22% (P = 0.0004). We identified three genome-wide significant associations with Dl_CO: variants near TGFB2, CHRNA3, and PDE11A loci (P < 5 × 10⁻⁸). In addition, 12 loci were suggestively associated with Dl_CO in European ancestry white (P < 1 × 10⁻⁵ in the combined analysis and P < 0.05 in both COPDGene and GenKOLS), including variants near NEGR1, CADM2, PCDH7, RETREG1, DACT2, NRG1, ANKRD18A, KRT86, NTN4, ARHGAP28, INSR, and PCBP3. Some Dl_CO-associated variants were also associated with COPD, emphysema, and/or spirometric values. Among 25 previously reported COPD loci, TGFB2, CHRNA3/CHRNA5, FAM13A, DSP, and CYP2A6 were associated with Dl_CO (P < 0.001). We identified several genetic loci that were significantly associated with Dl_CO and characterized effects of known COPD-associated loci on Dl_CO. These results could lead to better understanding of the heterogeneous nature of COPD.