A cohort study to identify incidence and risk factors of hookworm infection was conducted in a rural community, central Thailand from November 2005 to February 2007. Stool specimens were examined for hookworm eggs using wet preparation, Kato thick smear, and water-ethyl acetate sedimentation technique. The incidence rate of hookworm infection was 7.5/100 person-years. The independent risk factors for acquiring hookworm infection were barefoot walking (incidence rate ratio [IRR] = 4.2, 95% confidence interval [CI] = 1.2–14.5) and raising buffaloes around the house (IRR = 4.8, 95% CI = 1.9–11.8). Sequencing of internal transcribed spacer 1 (ITS1)-5.8S-ITS2 region of the ribosomal RNA gene were performed for identifying species of hookworm. Necator americanus was the most common hookworm identified in this population. Ancylostoma duodenale and A. ceylanicum were also detected. Our data suggest transmission of both human and animal hookworms in this community. Thus, prevention and control strategies of hookworm infection should cover both human and animal infection.