Blood culture-negative infective endocarditis (CNE) can be a diagnostic dilemma. Herein, we report a case of CNE caused by Haemophilus parainfluenzae identified only via 16S rRNA sequence analysis directly from valve tissue. A 17-year-old boy presented with high spiking fever for one month. Pansystolic murmur (Grade III) and vegetation (0.65×0.26 cm and 0.62×0.55 cm) on the anterior mitral valve leaflet via transesophageal echocardiogram suggested the diagnosis of infective endocarditis (IE). However, blood culture performed on admission was negative even after 2 weeks of incubation. Gram stain and culture of a direct tissue specimen failed to identify causative microorganism, while 16S rRNA gene sequences (548 bp) showed 100% identity with those of Haemophilus parainfluenzae (GenBank: FJ939586.1). The 16S rRNA sequence analysis with a direct tissue specimen might be useful in cases of CNE. (Korean J Clin Microbiol 2012;15:139-142)