Chronic kidney disease of unknown etiology (CKDu) is an epidemic that affects young agricultural workers in several warm regions of the world. However, there is a lack of monitoring of kidney issues in regions with extremely warm environments such as the Northwest of Mexico, a semi-arid region with a growing agricultural industry, where migrant and seasonal farm workers (MSFWs) travel to work in the fields. The objective of this study was to longitudinally assess kidney functioning of MSFWs in relation to pesticide exposure, heat stress and dehydration in a large-scale farm in Mexico. We enrolled 101 MSFWs, of whom 50 were randomly selected to work in an organic certified area and 51 were randomly selected to work in a conventional area. We also enrolled 50 office workers within the same region as a reference group. We collected urine and blood samples from all workers in addition to demographic, behavioral, and occupational characteristics. The physiological strain index (PSI) was used to estimate workers’ heat strain. Sampling was conducted at pre-harvest (March) and late in the harvest (July). Linear mixed models were built with the estimated glomerular filtration rate (eGFR) as the dependent variable. We found a significant decrease in kidney function in MSFWs compared to office workers. By the late harvest, one MSFW developed kidney disease, two MSFWs suffered a kidney injury, and 14 MSFWs were at risk of a kidney injury. We found that the eGFR in MSFWs decreased significantly from pre-harvest (125 ± 13.0 mL/min/1.73 m
2
) to late harvest (109 ± 13.6 mL/min/1.73 m
2
) (p < 0.001), while no significant change was observed in office workers. The eGFR was significantly lower in MSFWs who worked in the conventional field (101.2 ± 19.4 mL/min/1.73 m
2
) vs the organic field (110.9 ± 13.6 mL/min/1.73 m
2
) (p = 0.002). In our final model, we found that dehydration was associated with the decrease of eGFR. We also found an interaction between heat strain and job category, as a significant decline in eGFR by job category (conventional/organic MSFWs and office workers) was related to an increase in heat strain. This suggests that pesticide exposure needs to be considered in combination with heat stress and dehydration. This study provides valuable information on kidney function in MSFWs, and it shows the importance of early long-term monitoring in farm workers in other regions where CKDu has not been evaluated yet.