Carnation bacterial wilt (CBW), caused by Burkholderia caryophylli, is one of the most damaging diseases affecting carnations in Japan. In this study, carnation breeding was conducted using CBW resistance derived from Dianthus capitatus ssp. andrzejowskianus. To map the genetic loci involved in resistance to CBW and develop the linked markers, the first molecular linkage map for carnation was constructed by using 134 progeny derived from a cross between ‘Carnation Nou No. 1’ (an interspecific hybrid of carnation and D. capitatus) and ‘Pretty Favvare’, a susceptible cultivar. The map consisted of 146 DNA markers and covered 16 linkage groups. QTL analysis identified a QTL with a significant effect and two QTLs with small effects. Evaluation of disease incidence in relation to the presence of the STS-WG44 marker, which is linked to a QTL with a large effect, revealed that marker-assisted selection (MAS) using STS-WG44 enables the tested population to be narrowed down by half. Repeated crossing and selection via both conventional disease screening and MAS led to successful development of the first CBW-resistant carnation cultivar, ‘Karen Rouge’.