Bovine anaplasmosis is a tick-borne disease caused by Anaplasma marginale, although mechanical transmission by biting flies also occurs. Infection with A. marginale can reach 26% mortality and morbidity is associated with reduced beef and milk production, causing economic losses for livestock producers. Between March 2022 and July 2023, 1920 ticks were collected from 52 cattle production units in major cattle-producing states in Mexico, including Chiapas, Jalisco, Michoacán, Tabasco, and Veracruz. Of all the ticks collected, 35.57% were morphologically identified as Amblyomma mixtum. Samples of A. mixtum from each state, totaling 271, were tested for A. marginale via polymerase chain reaction (PCR). A. marginale was detected molecularly in 15.3% of A. mixtum samples. A. mixtum from Chiapas had the highest prevalence of A. marginale (24.0%), followed by Tabasco and Veracruz (20.0% each), Jalisco (15.2%), and Michoacán (6.6%). Phylogenetic analysis supported the morphological identification of A. mixtum and confirmed the genetic identity of A. marginale. This research is the first report on the molecular detection of A. marginale in A. mixtum ticks in Mexico. Results suggest that this 3-host tick species might be a potential vector. A. mixtum is Mexico’s second most economically significant tick feeding on cattle after Rhipicephalus microplus. This information about A. marginale prevalence in A. mixtum expands our understanding of bovine anaplasmosis epidemiology in Mexico. Further research is needed to assess the role of A. mixtum as a vector of A. marginale.