Over the last decade, erythromycin resistance has been increasing in frequency in Streptococcus pneumoniae in Denmark. In the present study, 49 non-related erythromycin-resistant S. pneumoniae isolates from invasive sites and 20 isolates from non-invasive sites were collected; antimicrobial susceptibility was tested, and they were genotyped and serotyped. Gene transfer was studied for selected isolates. The frequency of erm(B) was significantly higher in non-invasive isolates compared to invasive isolates (p = 0.001). For the first time, mef(I) was detected in 1 isolate in Denmark. All tested mef(E) isolates had an identical mef(E) sequence, apart from 1 gene with a point mutation, and mef(E) was correlated to 7 different sero-types. The tested erm(B) sequences were 99.3% similar with 5 point mutations at different positions distributed among different serotypes, which did not cause a detectable influence on the protein. Transformation was detectable in 5 out of 13 isolates and transfer of erm(B), mef(I) and mef(E) was detected. To our knowledge, this is the first time mef(I) has been proved transformable. Gene transfer by conjugation was not detectable. Erythromycin resistance in pneumococcal isolates is likely to be caused primarily by horizontal spread of mef(E) and erm(B), as well as clonal spread of a serotype 14 strain carrying mef(A) primarily detected in invasive isolates.