Megalospheric specimens of Heterostegina from four localities in Western and Central Cuba were morphometrically investigated using test characters, which are described by 15 growth-independent and growth-invariant attributes that enable complete test reconstruction in equatorial sections. The species Heterostegina cubana, H. ocalana, and Heterostegina sp. indet. were classified by nonmetric multidimensional scaling and cluster analysis. Discriminant analysis yielded significant separators between species such as the perimeter ratio of the first chamberlets, the decrease in chamberlet length from the previous spiral to the marginal side, the chamber height at the spiral side of the previous whorl, and the proloculus size. Based on a further discriminant analysis, specimens of H. ocalana from different localities, including specimens from Panama, are strongly separated by the number of operculinid chambers, the extension grade of the marginal spiral and the backward bend of chambers, documenting paleogeographic differences and apparent evolutionary trends such as the reduction of the number of operculinid chambers.

In Cuba, Heterostegina is represented by H. cubana and Heterostegina sp. indet. in the Bartonian to lower Priabonian (calcareous nannofossil Zones NP 16-NP 17), while Heterostegina ocalana ranges from Priabonian (nannofossil Zones NP 17 to NP 19-20/CP 15) to probably lower Oligocene (Rupelian) in the lower and middle part of the planktic foraminiferal Zone O 1 (P 18) and in the middle part of the calcareous nannofossil Zone NP 21 (CP 16).