Among the methods used for water quality monitoring in freshwater ecosystems we can find the phytotoxicity assessment and the seed germination and root elongation tests, which are the simplest methods recommended for environmental biomonitoring. The aims of this study were to assess the water toxicity of the Chalma river by the use of a bioassay with Lactuca sativa and to elaborate a biological index to diminish the water pollution condition of this river. Six study sites along the river were chosen, four of them located in the state of Mexico and two in the state of Morelos. The results were analyzed with multivariate methods: a principal component analysis and a similarity analysis. Three zones were differentiated according to the toxicity indices obtained: 1) the first three study sites showed positive values and low toxicity with high concentrations of nutrients, which could be promoting radical elongation, 2) the middle portion, represented only by the fourth study site, exhibited the lowest value of the root length index, with a classification of moderate toxicity and 3) the lower portion of the river, whose indices classified this zone as moderate to low toxicity. The last portion of the river was also associated with the accumulation of leachate compounds during the rainy season. Finally, the indices obtained showed that the sensitivity of the Lactuca sativa seeds bioassay generated an integral biological response. This information can be used for ecotoxicological risk assessment.