Carbon (δ13C) and nitrogen (δ15N) stable isotope analyses are used in marine ecology to study trophic relationships and migrations of species since they reflect dietary sources consumed which may vary geographically. However, better estimations of isotope incorporation rates and trophic discrimination factors (TDF) under controlled conditions are required. Moreover, variability of isotope incorporation rates and TDF among and within populations has been poorly described, especially in fish scales, whereas the use of non-lethal method is becoming a standard. This study aimed to experimentally assess whether carbon and nitrogen isotope incorporation rates (λC and λN, respectively) and TDF of scales vary in the European sea bass (Dicentrarchus labrax) among (1) Atlantic, West Mediterranean and East Mediterranean populations, (2) sexes and (3) individuals. Fish were reared under controlled conditions and switched from a diet 1 to a diet 2 with different δ13C and δ15N values. Scales were sampled repeatedly on 16 fish within the three populations, from the day of diet change (day 0) to the end of the experiment (day 217). Isotope incorporation rates of scales and TDF were determined using a time-dependent model. Isotopic carbon and nitrogen half-lives (t50C and t50N) were similar among the three populations but males had significantly lower t50C and t50N than females (29 ± 2 and 35 ± 2 days vs. 53 ± 7 and 80 ± 11 days, respectively). Females had higher growth rates but lower catabolic rates than males. Variability of λC and λN was large within sexes: t50C ranged from 17 to 159 days and t50N ranged from 18 to 342 days among individuals. Thus, variability between sexes and among individuals must be considered to avoid misinterpretation in field-based studies. For the 48 fish, TDF were 4.91 ± 0.03 and 2.46 ± 0.06‰ for carbon and nitrogen, respectively, and similar between sexes and among populations. Besides, TDF varied among individuals from 2.95 to 5.59‰ and from 0.93 to 3.55‰ for carbon and nitrogen, respectively. Empirical mixing models were run to estimate how different TDF influenced estimation of the contributions of food sources to diet of their consumer. The output differed considerably when using TDF from fish literature or those estimated herein, which confirms that a tissue-specific TDF must be used to avoid misinterpretation in field-based studies. Individual variation in TDF did not, however, influence estimation of the contributions of food sources, confirming that scales are a valid tissue for non-lethal sampling.