Colibacillosis is a chicken disease caused by avian pathogenic Escherichia coli (APEC). Pathogenicity in birds is determined by the occurrence of bacterial genes encoding virulence factors in APEC strains. Furthermore, APEC and other bacterial infections in commercial poultry farms have been treated with intensive use of antimicrobials for decades. Currently, many APEC strains are no longer susceptible to frequently used antibiotics due to increasing antimicrobial resistance (AMR) associated with the acquisition and mutation of other specific bacterial genes. The present study aimed to isolate and detect APEC isolates in broiler farms from different poultry-producing regions of Brazil and to determine their AMR profile. A total of 126 E. coli isolates were obtained from necropsied chickens with colibacillosis. All of these E. coli isolates were analyzed with one species-specific qPCR (targeting uspA gene) and five virulence factors genes qPCRs (targeting iroN, hlyF, iutA, iss, and ompT). AMR was determined by disk diffusion method using ten drugs frequently used to treat colibacillosis in Brazilian poultry farms. The results demonstrated that 109 (86.5%) isolates were classified as APEC. AMR was commonly observed in APEC and AFEC isolates, highlighting resistance for amoxicillin (85; 67.4%) and ceftiofur (72; 57.1%). A total of 41 (32.5%) E. coli isolates presented a multidrug resistance (MDR) profile. These results can contribute to implementing more effective colibacillosis prevention and control programs on Brazilian poultry farms.