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Despite the potential benefits gained from behavioural lateralisation, defined as the asymmetrical expression of cognitive functioning, this trait demonstrates widespread variation within and between populations. Numerous methodologies have been applied to investigate lateralisation, although whether different methodologies give consistent results has been relatively understudied. In this study, we assess (1) the repeatability of individual  Poecilia reticulata ’s lateralisation indexes between a classic detour assay (I-maze), quasi-circular mirror maze and novel detour assay (a radially symmetric Y-maze); (2) whether the methodological standard of analysing only the first ten turns in a detour assay accurately quantifies lateralisation; and (3) whether lateralisation indexes produced can be adequately explained by random chance by comparing the observed data to a novel unbiased ‘coin-toss’ randomisation model. We found (1) the two detour assays to produce generally consistent results in terms of relative lateralisation (directionality) but differed in terms of absolute laterality (intensity). The mirror assay, however, demonstrated no similarity to either assay. (2) The first ten turns were generally reflective of all turns undertaken during the 15-min trial but reducing the number of turns did exaggerate lateralisation indexes. (3) The observed laterality indexes from the assays were found to be similar to corresponding datasets produced by the randomisation model, with significant deviations likely explained by individuals’ propensity to perform consecutive turns in the same direction. These results demonstrate the need to increase the number of observed turning choices to reduce the likelihood of producing spurious or exaggerated lateralisation indexes from random chance or external influences. Significance statement Published studies investigating lateralisation, or ‘handedness’, in fish species have used a diverse array of methodologies. Given the variability in methodologies being employed and the widespread variation in the extent fish are lateralised and in which direction (left or right), it is important to assess whether different methods produce consistent laterality indexes. From assessing individual  Poecilia reticulata  in three laterality assays, the direction of lateralisation was found to correlate between the two detour assays measuring turn choice, although the absolute strength of this laterality was not consistent. There were no correlations between these assays and in an individual’s eye-use when viewing their reflection in a mirror maze assay. However, further investigation using a novel unbiased ‘coin-toss’ randomisation model to simulate replica datasets for each assay brings into question whether patterns of laterality found in the observed population differ significantly from random chance.

Detecting behavioural lateralisation in Poecilia reticulata is strongly dependent on experimental design