Rates of infection of Amblyomma americanum (L.) by Ehrlichia chaffeensis were compared in 100 ticks collected from sites in each of four states: Indiana, North Carolina, Kentucky, and Mississippi. The overall infection rates were similar among sites, ranging from 1 to 4%. Because pathogenic differences may exist between E. chaffeensis strains, nested polymerase chain reaction (PCR) amplification of the variable-length PCR target (VLPT), and sequencing of the amplicons were performed to differentiate between strains. The most common infecting strains at all sites exhibited a repeat profile of 1,2,3,4 (corresponding to the Arkansas/Jax/Osceola and Liberty strains). To determine whether the minimum infection rates (MIRs) or the most common infecting strain were changing over time in southern Indiana sites, 2,765 ticks from six counties in 2000 and 837 ticks from seven counties in 2004 also were examined in pools of five ticks per pool. The MIRs for 2000 and 2004 were 3.5 and 4.2% respectively, suggesting that the overall MIRs remained low. At two sites, in Pike and Harrison counties, however, infection rates more than doubled from 2000 to 2004 (7 to 16% and 0.3 to 2.7% respectively). Across all sites, the most common infecting strains (Arkansas/Jax/Osceola and Liberty) did not significantly change (68% in 2000; 79% in 2004).