We report the application of a recently developed molecular method, single step nested multiplex PCR (SSNM-PCR) assay and microscopy to identify and investigate temporal patterns of bivalve larvae in a Danish estuary, Isefjord. All samples were collected during the SUSTAINEX program from June to November 2001. Using the molecular assay, larvae could be categorized into six groups: the blue mussel, Mytilus edulis, Ensis spp., species of the Myoidae superfamily (Mya spp.), the common cockle (Cardiidae family), members of the Abra and Macoma genera of the Tellinoidae superfamily and members of the surf clam genera, Spisula spp. A seventh group was composed of unknown larvae. Greater resolution was possible by microscopy, but only for relatively large and intact individuals (>150–200 µm). The molecular approach was capable of differentiating between larvae regardless of shell size. Where it was possible to directly compare identifications based on both methods, concordance was high for M. edulis, Macoma balthica/Abra alba and E. americanus, whereas identification of Myoidae spp. and Cardiids was less consistent. Over the course of the study, two patterns of larval occurrence were observed. Larvae from species known to exhibit a protracted annual spawning period (M. edulis, Myoidae spp., Mysella bidentata and Cardiids) were present in the water column throughout the sampling period, whereas larvae of Abra alba, Barnea candida, E. americanus, Macoma balthica, Musculus marmoratus Scrobicularia plana and Tapes pullastra appeared at clearly defined periods.