Ciliates are unicellular eukaryotes with separate germline and somatic genomes and diverse life cycles, which make them a unique model to improve our understanding of population genetics through the detection of genetic variations. However, traditional sequencing methods cannot be directly applied to ciliates because the majority are uncultivated. Single-cell whole-genome sequencing (WGS) is a powerful tool for studying genetic variation in microbes, but no studies have been performed in ciliates. We compared the use of single-cell WGS and bulk DNA WGS to detect genetic variation, specifically single nucleotide polymorphisms (SNPs), in the model ciliate Tetrahymena thermophila. Our analyses showed that (i) single-cell WGS has excellent performance regarding mapping rate and genome coverage but lower sequencing uniformity compared with bulk DNA WGS due to amplification bias (which was reproducible); (ii) false-positive SNP sites detected by single-cell WGS tend to occur in genomic regions with particularly high sequencing depth and high rate of C:G to T:A base changes; (iii) SNPs detected in three or more cells should be reliable (an detection efficiency of 83.4-97.4% was obtained for combined data from three cells). This analytical method could be adapted to measure genetic variation in other ciliates and broaden research into ciliate population genetics.