The aim of this study was to evaluate the water quality in two streams of the Valles region of Jalisco, Mexico and fully determine if they are being used as tequila vinasse disposal sites. Three sampling campaigns were carried out at eight different points of the two streams that run near tequila factories (TFs). Different physicochemical parameters of water quality were measured: chemical oxygen demand (COD); biochemical oxygen demand (BOD5); total suspended solids (TSSs); total phosphates; fats, oils, and grease (FOG); Kjeldal nitrogen; nitrite; nitrate; pH; conductivity; temperature; dissolved oxygen (DO); and turbidity. Also, the analysis of samples of tequila vinasses (TVs) diluted with tap water were carried out to have a reference for the level of pollution in the streams. Furthermore, due to the fact that COD could be considered the main indicator of pollution with TVs, a linear regression was performed between COD concentrations and the percentage of dilution of TVs (with tap water). A positive correlation was found between these two variables, and based on this analysis, the vinasse content was estimated at each sampling point of the streams. It was found that on average, a volume of 8.5 ± 6.3% and 11.5 ± 4.9% of TVs were present in each sampling point of the Atizcoa and Jarritos Streams, respectively. Additionally, it was found that, in general, the concentration of pollutants increased as the streams passed through the TFs, particularly the Atizcoa Stream. According to the Water National Commission criteria, most of the points would be classified as highly polluted, since they reach concentrations of COD and BOD5 up to 6590 mg/L and 3775 mg/L, respectively, temperature values up to 37 °C, and DO values of 0.5 mg/L. Therefore, it was confirmed that the streams are being used as tequila vinasse disposal sites. Due to the above, there is an urgent need for tequila companies to implement treatment systems for the vinasse generated, since under current conditions, the monitored streams are practically devoid of aquatic life.