Neonatal diarrhea is a complex infectious disease caused by the interaction of adverse environmental conditions, deficient immune status of neonates and infection of enteropathogens. A variety of microorganisms can cause diarrhea in alpaca neonates, including bacteria, viruses and parasites. In the 1980s, isolation of Escherichia coli strains capable of inducing fluid accumulation in the intestinal loop assay in alpaca and llama neonates was described. However, virulence factors of E. coli associated with neonatal diarrhea in South American camelids (SAC) have been poorly characterised. In this study, 94 E. coli isolates from 94 crias of alpaca (1 per animal) with diarrhea were analysed for the presence of virulence factor genes of diarrheagenic E. coli by PCR. The investigated genes were verotoxin (vtl and vt2), intimin (eae), and bundle-forming pili (bfp) genes. The animals belonged to six different herds of the Andean region of Cuzco (Peru) and were between two and nine weeks old. Two categories of diarrheagenic E. coli strains were detected: Enteropathogenic E. coli (EPEC), and Verotoxin-producing E. coli (VTEC) strains. Out of the 94 analysed strains 2.13% (2/94) were EPEC (eae and bfp), 10.64% (10/94) were atypical EPEC (eae but bfp negative), and 11.7% (11/94) were VTEC. Only 3 of the VTEC strains (3/11) were eae positive: one eae vt2 and two eae vtl. All of the other VTEC strains (8/11) were only vtl. Infection with diarrheagenic E. coli strains was detected in all the herds. The results indicate that these pathogenic strains are circulating among domestic SAC, probably at high rates, in the Andean region of Peru. Therefore, EPEC and VTEC strains may be a leading cause of diarrhea in these animals. In addition, these pathogens are a matter for public health. Preventive measures based on vaccination and improvement of hygiene conditions are essential to reduce the impact of neonatal diarrhea in SAC health and mortality in the Andean region of Peru.