Millions of single nucleotide polymorphisms (SNP) are now known in the chicken, but the ones that segregate may differ among populations. The current study characterized a simple method to simultaneously identify SNP in populations and to estimate allele frequencies by using relative peak heights of nucleotides in sequencing traces from DNA pools. In the first experiment, known concentration gradients of alleles were produced by measured admixture of DNA from highly inbred individual chickens. In the second experiment, pools of DNA from 100 individuals of each of 3 populations were produced, and the estimated frequencies were compared with those from genotyping of individuals sampled from the same populations. Linear relationships (slope = 0.93 to 1.02) were demonstrated between relative peak heights of nucleotide sequence traces and allelic frequency from both types of experimental samples. The correlation of frequency estimates from pools and individual genotyping was 0.917 to 0.997. Therefore, relative peak heights of nucleotides in sequence traces from DNA isolated from blood pools provide accurate estimates of SNP frequencies of the population from which the pool individuals were sampled and can, therefore, increase efficiency of selecting SNP to genotype in specific populations.