Cancer stem cells (CSCs) are hypothesized to govern the origin, progression, drug resistance, recurrence and metastasis of human cancer. CSCs have been identified in nearly all types of human cancer, including esophageal squamous cell cancer (ESCC). Four major methods are typically used to isolate or enrich CSCs, including: i) fluorescence-activated cell sorting or magnetic-activated cell sorting using cell-specific surface markers; ii) stem cell markers, including aldehyde dehydrogenase 1 family member A1; iii) side population cell phenotype markers; and iv) microsphere culture methods. ESCC stem cells have been identified using a number of these methods. An increasing number of stem cell signatures and pathways have been identified, which have assisted in the clarification of molecular mechanisms that regulate the stemness of ESCC stem cells. Certain viruses, such as human papillomavirus and hepatitis B virus, are also considered to be important in the formation of CSCs, and there is a crosstalk between stemness and viruses-associated genes/pathways, which may suggest a potential therapeutic strategy for the eradication of CSCs. In the present review, findings are summarized along these lines of inquiry.