Abstract Background Dirofilaria immitis causes dirofilariosis, a potentially fatal condition in canids. Dirofilaria infections can be prevented with a macrocyclic lactone (ML) prophylactic regimen. However, some D. immitis isolates have become resistant to MLs. Genetic changes on the P-glycoprotein 11 gene, encoding an ABCB transporter, have been linked to the ML-resistant phenotypes and have been proposed as markers of drug resistance. However, nothing is known about the expression and the localization of this transporter in D. immitis , despite its strong link to ML-resistant phenotypes. Methods We examined the clinically validated D. immitis P-glycoprotein 11 ( Dim Pgp-11) single nucleotide polymorphism (SNP) via MiSeq analysis in three ML-susceptible isolates (Missouri, MP3 and Yazoo) and two ML-resistant isolates (JYD-34 and Metairie), and correlated the data with previously published MiSeq results of USA laboratory-maintained D. immitis isolates. The level of the expression of the Dim Pgp-11 messenger RNA transcript was analyzed by droplet digital PCR (ddPCR) and compared in the USA laboratory-maintained isolates, namely the ML-susceptible Missouri and Berkeley isolates, the putative ML-susceptible Georgia III and Big Head isolates and the ML-resistant isolate JYD-34. The immunolocalization of Dim Pgp-11 was visualized in the microfilaria (mf) life stage of the Missouri isolate using confocal microscopy. Results The results confirmed that the SNP found on Dim Pgp-11 is differentially expressed in the USA laboratory-maintained isolates. The ML-susceptible isolates had an alternate allele frequency of between 0% and 15%, while it ranged between 17% and 56% in the ML-resistant isolates. The constitutive expression of Dim Pgp-11 was similar in the Berkeley, Georgia III and Big Head isolates, while it was significantly decreased in the ML-resistant JYD-34 isolate ( P < 0.05), when compared to the ML-susceptible Missouri isolate. The Dim Pgp-11 protein was distinctly localized within the excretory-secretory (ES) duct, pore cells and the excretory cell and, more faintly, along the mf body wall. Conclusion Our data confirm that genetic polymorphism of Dim Pgp-11 is associated with ML resistance in USA laboratory-maintained D. imminits isolates. A link between Dim Pgp-11 and ML resistance in D. immitis is further supported by the lower protein expression in the ML-resistant JYD-34 isolate when compared with the ML-susceptible Missouri isolate. Interestingly, Dim Pgp-11 is strategically located surrounding the ES pore where it could play an active role in ML efflux. Graphical Abstract