Despite similar cellular distribution, individual TLRs induce distinct type I IFN subtype expression patterns that are associated with a preferential expression of selected IFN‐inducible genes. Human I‐IFNs include IFN‐β and 13 independently regulated subtypes of IFN‐α (I‐IFNs). TLR7 and ‐9 induce I‐IFNs, but it is unknown whether their subtype repertoire is similar. This study used new PCR arrays that selectively amplify individual I‐IFN subtype genes of human and nonhuman primates to characterize the TLR7‐ and ‐9‐mediated IFN response in vitro and in vivo. We show that in human PBMCs, TLR7 agonists induce a rapid burst of I‐IFN transcripts, consisting primarily of IFN‐α1/13, ‐α2, and ‐α14. In contrast, TLR9 agonists, regardless of the type used (CpG C‐, B‐, or D‐ODN), prompted slower but sustained expression of IFN‐α1/13, ‐α2, ‐α7, ‐α8, ‐α10, ‐α14, ‐α16, and ‐α21. These qualitative differences were translated downstream as differences in the pattern of IFN‐inducible genes. In macaque PBMCs, imiquimod produced a short burst of IFN mRNA, dominated by IFN‐α8, whereas C‐ or D‐ODN induced a greater than tenfold increase in transcripts for all I‐IFN subtypes by 12 h of culture. Differences were more evident in vivo, where TLR7 and ‐9 agonists induced significantly different levels of I‐IFN transcripts in skin. Although the rates of gene transcription differed significantly for individual TLR9 agonists, their IFN‐α subtype signature was almost identical, indicating that the type of receptor dictates the quality of the I‐IFN response in vitro and in vivo. These results may underlie the differential therapeutic effects of TLR7 and ‐9 agonists and should inform future clinical studies.