The phylogenetic relationships of the order Pleuronectiformes are controversial and at some crucial points remain unresolved. To date most phylogenetic studies on this order have been based on morpho-anatomical criteria, whereas only a few sequence comparisons based studies have been reported. In the present study, the phylogenetic relationships of 30 flatfish species pertaining to seven different families were examined by sequence analysis of the first half of the 16S mitochondrial DNA gene. The results obtained did not support percoids as the sister group of pleuronectiforms. The monophyletic origin of most families analyzed, Soleidae, Scophthalmidae, Achiridae, Pleuronectidae and Bothidae, was strongly supported, except for Paralichthyidae which was clearly subdivided into two groups, one of them associated with high confidence to Pleuronectidae. The analysis of the 16S rRNA gene also suggested the monophyly of Pleuronectiforms as the most probable hypothesis and consistently supported some major interfamily groupings.Las realaciones filogenétics del Orden Pleuronectiformes, son controvertidas, permaneciendo aun en algunos puntos esenciales en discusión. La mayoría de los estudios filogenéticos hechos hasta ahora han estado basados en criterios morfo-anatómicos y soilo unos pocos en la comparación de secuencias. En el presente estudio se han examinado 30 especies de peces planos pertenecientes a 7 familias distintas mediante la secuenciación de l primero mitad del gen 16S del ADN mitocondrial. El análisis de este gen apunta a la monofília del órden Pleuronectiformes.Peer reviewed