Escherichia coli is a major pathogen leading to systemic and enteric illnesses in wild giant pandas (Ailuropoda melanoleuca). To investigate the characteristics and distribution of E. coli in wild giant pandas across four different nature reserves in Sichuan, Republic of China, we researched serotypes, phylogenetic groups, antimicrobial resistance, and resistance genes of E. coli not previously reported for wild giant pandas. A total of 82 E. coli isolates were identified from 40 fecal samples in August 2016 to May 2017. The most-prevalent serogroups were O15 (4%, 3/82), O28 (2%, 2/82), and O44 (2%, 2/82). Antimicrobial resistance was highest for streptomycin (61%, 50/82) followed by amikacin (30%, 25/82). Among the four nature reserves, the proportion of streptomycin (86%, 12/14) and amikacin (57%, 8/14) was highest in Liziping. The frequencies of resistant genes aph(3′)-IIa, ant(3″)-Ia, aac(3)-IIa, aadA1, and StrB were 28, 23, 5, 21, and 32%, respectively, while none of the strains had the tetracycline gene. In Qianfoshan, the phylogenetic group B2 was the most common, comprising the highest percentage of isolates compared with the other seven phylogenetic groups. Furthermore, many variables such as phylogenetic groups, antimicrobial susceptibility, and resistance genes differed significantly (P<0.05) among the four nature reserves. In facilitating the safe discharge of captive giant pandas into the wild, as well as to support existing wild populations, the data from this research will prove invaluable to scientists and ecologists in their endeavors.