Enterovirus includes a diverse group of RNA-viruses that is part of the family of picornaviridae. Even though these viruses are distributed worldwide, there are some unpleasant illnesses they cause such as, minor respiratory tract infections and severe neurological diseases. The reason for this is the presence of extensive variability in the enterovirus genome and the ability of the like virus to spread without culture symptoms. Enteroviruses have been diagnosed since a long time using serological approaches but often encounter the drawbacks such as time-consuming tests, low-specificity, and low-sensitivity. The invention of polymerase chain reaction and next generation sequencing has fundamentally changed the approach to detection and typing of enteroviruses. They combine very high specificity together with very high sensitivity and this allows detecting of more than 10 enteroviruses including those that have a high mutation rate as well as those with low viral loads. However, co-existing infections are a significant problem because these diseases exhibit significant genetic diversity. This chapter aims to discuss about the fundamentals of enterovirus molecular diagnostics, highlighting important methods, their advantages, and their drawbacks. It also addresses the main difficulties in molecular diagnosis of enteroviruses and describes new strategies to get beyond these barriers.