Radioactive cDNAs to coxsackievirus B (CB) genomic RNAs were used to probe genomic homologies between RNAs of the CB group serotypes. CB5 shared approximately equivalent homology with the other five CB serotypes. CB1, CB2, and CB3 hybridized preferentially to CB5 in turn, suggesting that these three have diverged in one direction from CB5. CB4 and CB6 were more related to each other than to the other serotypes at a lower criterion, but at a more stringent criterion they did not demonstrate apparent homology with each other. This suggests that they have diverged from CB5 as well but perhaps earlier in time or at a greater rate than did CB1, CB2, or CB3. Some potential applications of these relationships to clinical CB detection assays based on nucleic acid hybridization are discussed.