Genomic DNAs extracted from 1,288 Haemaphysalis longicornis ticks collected from grass vegetation and various animals from nine provinces of Korea were subjected to screening by genus-specific (Ehrlichia spp. or Anaplasma spp.) real-time TaqMan PCR and speciesspecific (E. chaffeensis) nested-PCR based on amplification of 16S rRNA gene fragments. In all, 611 (47.4%) ticks tested positive for genus-specific amplification of 116 bp fragment of 16S rRNA of Ehrlichia spp. or Anaplasma spp. Subsequently, 396 bp E. chaffeensis-specific fragment of 16S rRNA was amplified from 4.2% (26/611) tick samples. The comparison of the nucleotide sequence of 16S rRNA gene from one tick (EC-PGHL, GeneBank accession number AY35042) with the sequences of 20 E. chaffeensis strains available in the database showed that EC-PGHL was 100% identical or similar to the Arkansas (AF416764), the Sapulpa (U60476) and the 91HE17 (U23503) strains. The phylogenetic analysis also revealed that the E. chaffeensis EC-PGHL formed a single cluster with the above strains. This is the first study to report molecular detection and phylogenetic analysis of E. chaffeensis from H. longicornis ticks in Korea. The implicit significance of E. chaffeensis infection in H. longicornis ticks in Korea is discussed.