Visualization of tissue and cellular architecture is essential for understanding the pathobiology of tick-borne virus infection. RNA in situ hybridization (ISH) utilizes specific oligonucleotide probes to detect the presence and specific location of complementary RNA transcript targets within a tissue section. Chromogenic or fluorescent staining technologies allow researchers to visualize multiple RNA targets within a single tissue. Herein, we describe an RNA ISH methodology based on a commercially available kit to hybridize specific probes to POWV RNA within infected tissues.