In this study, we determined the efficiency of electroejaculation and assessed sperm quality using conventional and advanced reproductive biomarkers following the extraction of spermatophores by either dissection or electroejaculation from wild-caught redclaw crayfish. In Experiment 1, sexually mature male crayfish (> 40 g) were collected from Ross River Dam, northern Queensland and were subjected to electroejaculation to evaluate the efficiency of electrical stimulation to extrude spermatophores. In Experiment 2, 33 male crayfish weighing 99.4 ± 7.6 g (mean ± SEM) were subjected to sperm quality assessment following isolation from spermatophores collected by dissection and electroejaculation. Body weights (p = 0.029) affected the odds of extracting spermatophore and were greatest within crayfish weighing between 60 and 130 g. The success rate in extracting spermatophores from either gonopore using electroejaculation was 22.2 % (n = 153). The mean weight of spermatophores from left (0.016 ± 0.002 g) or right (0.011 ± 0.002 g) gonopores were comparable and were not significantly influenced by body weight (p = 0.430). A greater mean concentration of spermatozoa, sperm viability, total potential fertile sperm concentration (TPFSC) and lesser sperm DNA fragmentation were measured following the extraction of spermatophores by dissection than those by electroejaculation (p < 0.001). Together, these data suggest that the method used for spermatophore collection impacts sperm quality in redclaw crayfish. Spermatophores collected following dissection provided better sperm quality and are recommended for developing advanced breeding techniques, including sperm freezing and artificial reproduction techniques. However, electroejaculation is a simple, non-lethal alternative for collecting spermatophores for routine breeding and commercial production of redclaw.