Sequence analysis provides a valuable alternative to phenotypic identification of moulds, especially for isolates lacking characteristic morphology. In this comparative prospective study, isolates that could not be identified by standard phenotypic criteria within 5 days (n = 244) were subjected to sequence analysis and further in-depth phenotypic investigations. Comparison of sequence-based with extended phenotypic identification revealed that sequence analysis was more precise in 52.0% of the isolates; in 38.6% of the isolates, both methods gave concordant results. The construction of a database consisting of high-quality sequences allowed improvement of sequence-based identification. Based on these results, we propose a diagnostic algorithm for the effective use of both phenotypic and genetic procedures for identification of moulds in the diagnostic laboratory.