Secondary and tertiary RNA structures play key roles in genome replication of picornaviruses. Complex, functional structures are particularly abundant in the untranslated regions, where they are involved in initiation of translation, priming of new strand synthesis and genome circularisation. The 5′ UTR of foot-and-mouth disease virus (FMDV) is predicted to end in a c. 360 nucleotide-long stem-loop, termed the short (S) fragment. This structure is highly conserved and essential for viral replication, but the precise function(s) is unclear. Here, the validity of earlier structural predictions was strengthened by comparative genomic analyses that confirmed structure conservation of S fragments from a wide range of field isolates. In addition, we used selective 2′ hydroxyl acetylation analysed by primer extension (SHAPE) to experimentally-determine the organisation of the structure of the genome. To examine the role of S fragment stem-loop structure in virus replication, we introduced a series of deletions to the distal and proximal regions. These truncations affected genome replication in a size-dependent and, in some cases, host cell-dependent manner. Furthermore, during passage of viruses incorporating the largest tolerated deletion from the proximal region of the S fragment stem-loop, an additional mutation was selected in the viral RNA-dependent RNA polymerase, 3Dpol, which influenced the function of the enzyme. These data suggest that the S fragment and 3Dpol interact in the formation of the FMDV replication complex.