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Anaplasmosis is increasingly common in the United States, with cases being reported over an expanding geographic area. To monitor for changes in risk of human infection, the U.S. Centers for Disease Control and Prevention monitors the distribution and abundance of host-seeking vector ticks (Ixodes scapularis and Ixodes pacificus) and their infection with Anaplasma phagocytophilum. While several variants of A. phagocytophilum circulate in I. scapularis, only the human-active variant (Ap-ha) appears to be pathogenic in humans. Failure to differentiate between human and non-human variants may artificially inflate estimates of the risk of human infection. Efforts to differentiate the Ap-ha variant from the deer variant (Ap-V1) in ticks typically rely on traditional PCR assays coupled with sequencing of PCR products. However, laboratories are increasingly turning to Next Generation Sequencing (NGS) to increase testing efficiency, retain high sensitivity, and increase specificity compared with traditional PCR assays. We describe a new NGS assay with novel targets that accurately segregate the Ap-ha variant from other non-human variants and further identify unique clades within the human and non-human variants. Recognizing that not all investigators have access to NGS technology, we also developed a PCR assay based on one of the novel targets so that variants can be visualized using agarose gel electrophoresis without the need for subsequent sequencing. Such an assay may be used to improve estimates of human risk of developing anaplasmosis in North America., (Copyright © 2022. Published by Elsevier GmbH.)