A convenience sampling of fecal specimens from 40 cats in northern Florida was examined for the presence of Ancylostoma braziliense eggs by using centrifugal sugar flotation and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP). Of the 40 samples, 26 (65%) contained hookworm eggs. DNA from 24 samples was successfully amplified using PCR; using RFLP, 10 samples were identified as containing DNA of A. braziliense (41.7% of the 24 samples that successfully amplified). Of these, 6 samples contained DNA of both Ancylostoma tubaeforme and A. braziliense, and 4 samples contained only DNA of A. braziliense. The remaining samples (n = 14) contained only the DNA of A. tubaeforme, except for 1 sample that had no discernible bands after RFLP.