The development of increasingly safe and effective antiretroviral treatments for human immunodeficiency virus (HIV) over the past several decades has led to vastly improved patient survival when treatment is available and affordable, an outcome that relies on uninterrupted adherence to combination antiretroviral therapy for life. Looking to the future, the discovery of an elusive ‘cure’ for HIV will necessitate highly sensitive methods for detecting, understanding, and eliminating viral reservoirs. Next-generation, in situ hybridization (ISH) approaches offer unique and complementary insights into viral reservoirs within their native tissue environments with a high degree of specificity and sensitivity. In this review, we will discuss how modern ISH techniques can be used, either alone or in conjunction with phenotypic characterization, to probe viral reservoir establishment and maintenance. In addition to focusing on how these techniques have already furthered our understanding of HIV reservoirs, we discuss potential avenues for how high-throughput, next-generation ISH may be applied. Finally, we will review how ISH could allow deeper phenotypic and contextual insights into HIV reservoir biology that should prove instrumental in moving the field closer to viral reservoir elimination needed for an ‘HIV cure’ to be realized.