Sperm quality, an important male fitness trait, is commonly compared between studies. However, few studies consider how genetic and environmental variation affect sperm quality, even in the genetic model Drosophila melanogaster. Here we show that sperm viability, the proportion of live sperm, differed across the genotypes Oregon-R, Dahomey, and Canton-S by more than 15%, and across buffers (phosphate-buffered saline (PBS), Grace's Medium and Drosophila Ringer solution) by more than 20%. In terms of genotype-buffer pair comparisons, nearly half of the comparisons would produce significant differences in sperm viability (15 in 36), or its temporal decrease in a stress medium (19 in 36). Grace's medium produced the longest-lived sperm in vitro and the smallest differences between genotypes, Drosophila Ringer Solution produced the shortest lifespan and the largest differences. Our results suggest that fly and other sperm researchers would benefit from a standardized protocol of measuring sperm viability.