Sequences at the beginnings and ends of Human respiratory syncytial virus (HRSV) genes are necessary for efficient initiation and termination of transcription. The gene start sequences are well conserved and contain signals required for initiation, while the semi-conserved sequences at the gene ends direct transcriptional termination with varying efficiencies. The intergenic regions, which lie between the gene ends and the downstream gene start sequences, are not conserved in length or sequence, and certain positions have been reported to play a role in transcriptional regulation. We have previously shown that the gene end sequences in HRSV subgroup A clinical isolates are variable and that variations found at certain gene ends decreased transcriptional termination and downstream mRNA expression. Here, we have extended this work to examine variation in the intergenic regions between the genes of clinical isolates. We determined the sequences of the eight intergenic regions and the M2/L overlap from clinical isolates from the US and UK and found that all of these regions contained variations from the prototype A2 strain. The amount of variation observed was disparate among the different intergenic regions and did not correlate with length. The effects of selected variant sequences on transcription were examined in the context of subgenomic replicons. While some changes in the intergenic regions had minor effects, certain sequence variations significantly altered transcription termination or initiation. A single nucleotide deletion in the M/SH intergenic region decreased initiation at the SH gene start seven-fold, while changes in the F/M2 intergenic region were found that in some cases increased and in others decreased termination at the F gene end. The P/M intergenic region was the most variable, but none of the changes examined affected either termination at the P gene end or initiation of the downstream M gene start. These results show that in HRSV clinical isolates the intergenic region sequences are variable and that changes in these regions have the potential to affect transcriptional control at the gene junctions.