Cytogenetic and morphometric analyses were carried out in Hoplias aff. malabaricus specimens from six distinct populations from the lower Paranapanema River basin, located between the states of Paraná and São Paulo, Brazil. Measurements were also taken from a specimen collected in Surinam. In a population from a fish farm station at Universidade do Norte do Parana (EPUNOPAR), two sympatric cytotypes (2n = 40 and 2n = 42 chromosomes) are found. A population from a fish farm station at Universidade Estadual de Londrina (EPUEL) shows 2n = 42 meta-submetacentric chromosomes for males and females with a simple sex chromosome system of XX/XY type. Populations from the Vermelho and Rancho Alegre Rivers, Três Bocas Stream and Paranapanema River have 2n = 39 chromosomes in males and 2n = 40 chromosomes in females, showing a multiple sex chromosome system of X(1)X(1)X(2)X(2)/X(1)X(2)Y type. Twenty morphological variables were studied. These measurements were used for an analysis of the canonical variables and standard analysis of proportional measurements. The most variable measurements among the specimens are the maxilla length (MXL) and the pre-dorsal distance (PDD). Analysis of canonical variables indicates three distinct groups in the first canonical axis formed by: (1) Três Bocas Stream, (2) Rancho Alegre + Vermelho River + EPUNOPAR and (3) EPUEL + Paranapanema River. This axis retained 79.4% of information from the original matrix. Analysis of morphometrics reveals differences among populations from the Paranapanema River basin and between these and the specimen from Surinam. The morphometric and cytogenetic differences among the studied populations suggest a species complex.