Abstract:  A retrospective study to detect Anaplasma phagocytophilum antibodies by indirect immunofluorescence (IFA) and Western blot (WB) assay was conducted in 367 potentially exposed patients from Portugal. The study included 26 patients with confirmed Lyme borreliosis (LB), 77 with suspected LB, 264 seronegative patients studied for possible tick‐transmitted LB and boutonneuse fever (LB/BF) infection, and 96 healthy blood donors. Overall, patients with LB and suspected LB (n= 2 [7.7%] and n= 6 [7.8%], respectively) were more often seropositive (n= 8 [7.8%]; P < 0.001), whereas only 1 (0.4%; P= 0.046) patient in the LB/BF seronegative group had confirmed disease. This study is the first evidence of human exposure to A. phagocytophilum or an antigenically similar bacterium in Portugal, and suggests that LB patients are significantly more likely to contact A. phagocytophilum.