Difflugia is a morphologically diverse genus of testate amoebae, which are common components of freshwater ecosystems. We observed a new morphotype similar to Difflugia tuberspinifera but without spine in four Xiamen reservoirs, Fujian Province, southeast China. We investigated its morphology and biometry using light and scanning electron microscopy. The linear discriminant analysis and principal component analysis of biometric characters revealed that the spiny and spineless forms of D. tuberspinifera differed only in the presence or absence of spine. Shell height, shell diameter, aperture diameter and collar height did not differ significantly between the two morphotypes. The number of conical spines varies from 0 to 8. However, the distribution of spine numbers showed two main modes at 0 (spineless form 45.1% of individuals) and 4-6 (38.9%), suggesting the possible existence of two genetically distinct lineages. Spines may have ecological and evolutionary significance. Our results suggested that the spiny and spineless morphotypes of D. tuberspinifera represent either a single variable taxon with different ecotypes or sibling species. Further morphological studies on clonal variations and molecular approaches are needed to clarify if the spineless morph represents an independent species or not.