Haemaphysalis longicornis (H. longicornis) is the most dominant species that accounts for more than 90% of domestic ticks in Korea. It is also a main mediator and propagator of Severe Fever with Thrombocytopenia Syndrome virus. In order to efficient control of H. longicornis, it is essential to obtain the genetic information of pesticide target genes and determine the mutations putatively involved in insecticide resistance. Acetylcholinesterase (Ace) is a target gene of organophosphorus and carbamate insecticides, but no sequence information of Ace is available to date in H. longicornis. Therefore, in this study, we analyzed the transcriptome of H. longicornis and obtained the sequence information of three Ace genes (HlAce1, HlAce2 and HlAce3). Based on the comparison analysis with sequences of multiple ace genes from other hard ticks, mites and insects, three HlAces were grouped in Acari Ace1, Ace2 and Ace3 clade, respectively. Among three HlAces, HlAce1 seems to be major enzyme because of its conserved amino acids involved in catalytic function.