Astroviruses are small, positive-sense single-stranded RNA viruses that cause gastroenteritis and, in some cases, neurological disease. Despite their significance, many aspects of their replication and RNA regulation remain unclear.  Through structural probing and computational modeling, we identified several conserved RNA elements within astrovirus genomes, including a distinct structured region located in the subgenomic RNA domain. This region, termed the subgenomic stem-loop (SG-SL), displayed strong evidence of functional importance based on sequence conservation and structural stability.  Using a replicon system that allows quantitative assessment of viral RNA replication, we performed targeted mutagenesis within the SG-SL. Several mutations in this region led to a marked reduction in replication efficiency, indicating that the RNA structure plays a crucial regulatory role. Analysis of these findings led us to hypothesize that the SG-SL may be directly involved in controlling the subgenomic RNA synthesis, for which we propose a potential mechanism.  Overall, our study identifies a key structured RNA element in astrovirus genomes and provides new insight into how RNA structure may regulate viral transcription mechanisms.