Tomato seedlings damping-off is a limiting factor in commercial greenhouse production. To determine the causal agents of disease, sampling and fungal isolation were performed during 2012. Samples were collected from infected seedlings growing in greenhouses in the Syrian coastal region. Isolation of fungi was done in the laboratories of the Agronomical Reaserch Center, in Lattakia and the molecular analyses were done in the Biotechnology Center at Tishreen University, Lattakia, Syria, during the years 2012, 2013. Eight isolates of Pythium sp. obtained were purified using hyphal tip method (named P1, P2, P3, P4, P5, P6, P7 and P8). Isolates were morphologically identified by optical microscope, then molecularly Characterized using genus specific ITS primers. The results of morphological characterization of pathogenic species suggested the detection of Pythium aphanidermatum, P. ultimum. The analysis of DNAs from the different isolates with ITS primers, recognizing the inter transcript spacer of nuclear ribosomal DNA proved that the eight, isolates were belonging to the species P. ultimum. The complete sequences of ribosomal DNA internal transcribed spacers regions of selected isolates were determined and submitted to GenBank. The GenBank-BLAST homology search revealed P. ultimum as the most similar sequence (> 96% identity) with GenBank entry AB355596.