An increase in melioidosis cases compared to other areas in Taiwan was observed in the Er-Ren River Basin, southwestern Taiwan, from November 2001 to August 2006. The objective of this study was to determine the association between the level of exposure to Burkholderia pseudomallei and the incidence rate of melioidosis and to survey the transmission modes of B. pseudomallei in the Er-Ren River Basin. The serosurveillance of melioidosis gave seropositivity rates of 36.6%, 21.6%, and 10.9%, respectively, for residents in regions A, B, and C within the Er-Ren Basin area. Culture and PCR-based detection of B. pseudomallei from soil demonstrated that the geographical distribution of this bacterium was confined to a particular site in region B. The distribution of seropositive titers was significantly associated with the incidence rate of melioidosis (120, 68, or 36 incidence cases per 100,000 population in region A, B, or C in 2005), whereas it did not correlate with the geographical distribution of B. pseudomallei within the soil. A survey of transmission modes showed that residents with seropositivity were linked to factors such as having confronted flooding and having walked barefoot on soil, which are potential risk factors associated with exposure to B. pseudomallei. Our findings indicated that the Er-Ren River Basin in Taiwan has the potential to become a high-prevalence area for melioidosis. This is the first report that documents a high prevalence of melioidosis in an area north of latitude 20 degrees N.