Large-scale population phenotyping for molecular epidemiological studies is subject to all the usual criteria of analytical chemistry. As part of a major phenotyping investigation we have used high-resolution 1H NMR spectroscopy to characterize 24-h urine specimens obtained from population samples in Aito Town, Japan (n = 259), Chicago, IL (n = 315), and Guangxi, China (n = 278). We have investigated analytical reproducibility, urine specimen storage procedures, interinstrument variability, and split specimen detection. Our data show that the multivariate analytical reproducibility of the NMR screening platform was >98% and that most classification errors were due to urine specimen handling inhomogeneity. Differences in metabolite profiles were then assessed for Aito Town, Chicago, and Guangxi population samples; novel combinations of biomarkers were detected that separated the population samples. These cross-population differences in urinary metabolites could be related to genetic, dietary, and gut microbial factors.