Much research has investigated the relationship between the Approximate Number System (ANS) and mathematical achievement, with continued debate surrounding the existence of such a link. The use of different stimulus displays may account for discrepancies in the findings. Indeed, closer scrutiny of the literature suggests that studies supporting a link between ANS acuity and mathematical achievement in adults have mostly measured the ANS using spatially intermixed displays (e.g. of blue and yellow dots), whereas those failing to replicate a link have primarily used spatially separated dot displays. The current study directly compared ANS acuity when using intermixed or separate dots, investigating how such methodological variation mediated the relationship between ANS acuity and mathematical achievement. ANS acuity was poorer and less reliable when measured with intermixed displays, with performance during both conditions related to inhibitory control. Crucially, mathematical achievement was significantly related to ANS accuracy difference (accuracy on congruent trials minus accuracy on incongruent trials) when measured with intermixed displays, but not with separate displays. The findings indicate that methodological variation affects ANS acuity outcomes, as well as the apparent relationship between the ANS and mathematical achievement. Moreover, the current study highlights the problem of low reliabilities of ANS measures. Further research is required to construct ANS measures with improved reliability, and to understand which processes may be responsible for the increased likelihood of finding a correlation between the ANS and mathematical achievement when using intermixed displays.