Calonectria represents a genus of phytopathogenic ascomycetous fungi with a worldwide distribution. In recent years, there has been an increase in the number of taxonomic studies on these fungi. Currently, there are 169 described species of Calonectria based on comparisons of DNA sequence data, combined with morphological characteristics. However, for some of these species, the sequence data utilised at the time of their description were relatively limited. This has justified an urgent need to reconsider the species boundaries for Calonectria based on robust genus-wide phylogenetic analyses. In this study, we utilised 240 available isolates including the ex-types of 128 Calonectria species, and re-sequenced eight gene regions (act, cmdA, his3, ITS, LSU, rpb2, tef1 and tub2) for them. Sequences for 44 Calonectria species, for which cultures could not be obtained, were downloaded from GenBank. DNA sequence data of all the 169 Calonectria species were then used to determine their phylogenetic relationships. As a consequence, 51 species were reduced to synonymy, two new species were identified, and the name Ca. lauri was validated. This resulted in the acceptance of 120 clearly defined Calonectria spp. The overall data revealed that the genus includes 11 species complexes, distributed across the Prolate and Sphaero-Naviculate Groups known to divide Calonectria. The results also made it possible to develop a robust set of DNA barcodes for Calonectria spp. To accomplish this goal, we evaluated the outcomes of each of the eight candidate DNA barcodes for the genus, as well as for each of the 11 species complexes. No single gene region provided a clear identity for all Calonectria species. Sequences of the tef1 and tub2 genes were the most reliable markers; those for the cmdA, his3, rpb2 and act gene regions also provided a relatively effective resolution for Calonectria spp., while the ITS and LSU failed to produce useful barcodes for species discrimination. At the species complex level, results showed that the most informative barcodes were inconsistent, but that a combination of six candidate barcodes (tef1, tub2, cmdA, his3, rpb2 and act) provided stable and reliable resolution for all 11 species complexes. A six-gene combined phylogeny resolved all 120 Calonectria species, and revealed that tef1, tub2, cmdA, his3, rpb2 and act gene regions are effective DNA barcodes for Calonectria.