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Lice are obligate ectoparasites of birds and mammals with specialized mouthparts adapted to feed on the blood or other body tissues of their respective hosts. The registry of parasites that can cause economic and health impacts on the buffalo herd of the country is of utmost importance. In the present study, we report the first record of Haematopinus tuberculatus parasitizing buffalo in the municipality of Autazes, Amazonas, Brazil. The study was conducted in a rural private area located on the banks of the Paraná Madeirinha River. Twelve specimens were collected and identified as H. tuberculatus, comprising five females, six males, and one third instar nymph. The lice were observed on females buffalo during milking; however, the greatest abundance was found on calves, in the neck, back, and loin regions. There was no visible DNA amplification on agarose gel for the samples tested. However, the record of this louse species parasitizing on buffaloes in the region highlights the potential for epizootic outbreaks to occur in the area.