In humans, rotavirus infection is potentially fatal, with approximately 128,000 deaths annually. Rotaviruses have an eleven-segmented double-stranded RNA genome which must undergo stoichiometric assortment of distinct RNA segments during replication. Multiplexed RNA FISH analysis suggests that all eleven RNAs accumulate in viroplasms, which are biomolecular condensates essential for rotaviral replication, yet the molecular basis of selective RNA packaging remains poorly understood. While sequence-specific protein–RNA interactions are well characterised, accumulating evidence points to a critical role for RNA–RNA interactions in driving segment assortment. To define these interactions, we applied RNA crosslinking approaches capable of capturing intermolecular RNA contacts, to enable us to dissect the impact of co-transcriptional folding and to compare RNA interaction networks formed by in vitro transcription versus those formed during transcription from isolated viral particles. We have identified key regions involved in forming inter-segment interactions co-transcriptionally, inducing conformational changes in RNA structure. By altering the RNA sequences involved in these long-range interactions using the established reverse genetics system for rotavirus we aim to elucidate contributory factors to packaging and ultimately the fate of viral RNA during infection.  Together, these approaches provide new insights into the mechanisms of segment selection and packaging in rotavirus, with broader relevance to the assembly of other segmented RNA viruses.