Human rhinoviruses (RVs), comprising three species (A, B and C) of genus Enterovirus, are responsible for the majority of upper respiratory tract infections and associated with severe lower respiratory tract illnesses such as pneumonia and asthma exacerbations. High genetic diversity and continuous identification of new types require regular updating of the diagnostic assays for accurate and comprehensive detection of circulating RVs. Methods for molecular typing based on phylogenetic comparisons of a variable fragment in the 5' untranslated region were improved to increase assay sensitivity and to eliminate non-specific amplification of human sequences observed occasionally in clinical samples. A modified set of primers based on new sequence information and improved buffers and enzymes in semi-nested PCR provided higher specificity and sensitivity of virus detection. In addition, new diagnostic primers were designed for unequivocal species and type assignment of RV-C isolates based on phylogenetic analysis of partial VP4/VP2 coding sequence. The improved assay was evaluated by typing RVs in more than 3,800 clinical samples. RV was successfully detected and typed in 99% of the samples that were RV-positive by multiplex diagnostic assays.