Emerging and re-emerging vector-borne infections are a global public health threat. In endemic regions, fever is the main reason for medical attention, and the etiological agent of such fever is not usually identified. In this study, non-specific febrile pathogens were molecularly characterized in serum samples from 253 patients suspected of arbovirus infection. The samples were collected in the southern border region of Mexico from April to June 2015, and February to March 2016. ZIKV, CHIKV, DENV, leptospirosis, and rickettsiosis were detected by qPCR and nested PCR to identify flavivirus and alphavirus genera. The results indicated that 71.93% of the samples were positive for CHIKV, 0.79% for ZIKV, and 0.39% for DENV, with the number positive for CHIKV increasing to 76.67% and those positive for ZIKV increasing to 15.41% under the nested PCR technique. Leptospira Kmetyi was identified for the first time in Mexico, with a prevalence of 3.16%. This is the first report of ZIKV in Mexico, as well the first detection of the virus in early 2015. In conclusion, the etiological agent of fever was determined in 94% of the analyzed samples.