In Lake Ladoga (northwestern Russia), we found a diatom, putatively Fragilaria sublanceolata-baikali, an endemic species from Lake Baikal (southeastern Siberia, Russia). To determine whether this population matches a previously recognized species from Lake Baikal and assess how it differs from other similar Fragilaria taxa, we studied the valve morphology of three morphologically similar Fragilaria populations (the putative F. sublanceolata-baikali, F. pectinalis and F. perminuta) sampled in Lake Ladoga, along with a population of F. sublanceolata-baikali sampled in Lake Baikal. We used light and scanning electron microscopy with a combination of traditional and geometric morphometric methods. To analyze covariation between the valve shape and size (i.e., allometry), we examined differences in the ontogenetic–allometric trajectories at both the interspecific and intraspecific levels. In addition, the effect of size correction of the valve shape on species differentiation was tested. Traditional morphometrics revealed that F. sublanceolata-baikali is distinguished from F. pectinalis and F. perminuta by valve length, while F. pectinalis and F. perminuta are distinguished by striae density. All three species of Fragilaria showed separate and parallel allometric trajectories. In contrast, the two populations of F. sublanceolata-baikali were on a common allometric trajectory, indicating the conspecificity between these populations. Prior to allometric correction, geometric morphometrics was not able fully discriminate between the three Fragilaria species. After allometric correction, the three Fragilaria species were clearly separated in a size-corrected morphospace, whereas the two populations of F. sublanceolata-baikali formed a tightly overlapping group. Thus, we conclude that geometric morphometrics can reliably distinguish between these morphologically similar species of Fragilaria, but only after accounting for allometric shape variation. Our study confirmed morphological similarity between the two geographically distant populations of F. sublanceolata-baikali, which indicates that this taxon can be considered as invasive in Lake Ladoga.