Wenlock–Ludlow boundary beds exposed near Nesvačily in the Prague Synform are documented in terms of their lithology, faunal content, high-resolution record of 19 graptolite species, graptolite biostratigraphy, and conodont record. This is the first time in Bohemia and peri-Gondwanan Europe that a continuous graptolite-bearing section through upper ludensis – middle nilssoni biozones is described in detail to refine high-resolution biostratigraphy and correlation of the Wenlock–Ludlow boundary. Organic-rich shale, relatively rare nongraptolite fauna, and absence of bioturbation indicate a stable deep-water offshore setting with sporadic, low-level bottom oxygenation. The uninterrupted succession revealed a series of origination events of principal graptolite taxa employed in determination of the Wenlock–Ludlow boundary strata worldwide. Bohemograptus represented by B. praecox appeared as early as in the uppermost Wenlock ludensis Biozone. Bohemograptus praecox is later accompanied and replaced by B. bohemicus, associated with Uncinatograptus uncinatus. The lowest Neodiversograptus nilssoni marked the base of the Ludlow nilssoni Biozone. Speciation within Bohemograptus continued in the middle nilssoni Biozone when the robust B. butovicensis evolved from B. bohemicus. Colonograptus colonus shows its lowest occurrence in the middle part of the nilssoni Biozone together with the latest C. gerhardi. The monograptids are associated with abundant and diverse plectograptids that are at present mostly known from Baltica. Bohemograptus praecox sp. nov. and B. butovicensis (Bouček, 1936) are described in the systematic part. The conodont Kockelella ortus absidata, found in the lower–middle part of the nilssoni Biozone, is consistent with a stratigraphic interval within Oz. bohemica and K. crassa conodont biozones.